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Catalytic and redox properties of glycine oxidase from Bacillus subtilis
Biochimie 91 (2009) 604–612
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Research paper
Catalytic and redox properties of glycine oxidase from Bacillus subtilis Mattia Pedotti, Sandro Ghisla, Laura Motteran, Gianluca Molla, Loredano Pollegioni* Department of Biotechnology and Molecular Sciences, University of Insubria, via J.H. Dunant 3, 21100 Varese, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 December 2008 Accepted 18 February 2009 Available online 28 February 2009
Glycine oxidase (GO) from Bacillus subtilis is a homotetrameric flavoprotein oxidase that catalyzes the oxidation of the amine functional group of sarcosine or glycine (and some D-amino acids) to yield the corresponding keto acids, ammonia/amine and H2O2. It shows optima at pH 7–8 for stability and pH 9–10 for activity, depending on the substrate. The tetrameric oligomeric state of the holoenzyme is not affected by pH in the 6.5–10 range. Free GO forms the anionic red semiquinone upon photoreduction. This species is thermodynamically stable, as indicated by the large separation of the two single-electron reduction potentials (DE 290 mV). The first potential is pH independent, while the second is dependent. The midpoint reduction potential exhibits a 23.4 mV/pH unit slope, which is consistent with an overall two-electrons/one-proton transfer in the reduction to yield anionic reduced flavin. In the presence of glycolate (a substrate analogue) and at pH 7.5 the potential for the semiquinone-reduced enzyme couple is shifted positively by w160 mV: this favors a two-electron transfer compared to the free enzyme. Binding of glycolate and sulfite is also affected by pH, showing dependencies that reflect the ionization of an active site residue with a pKa z 8.0. These results highlight substantial differences between GO and related flavoenzymes. This knowledge will facilitate biotechnological use of GO, e.g. as an innovative tool for the in vivo detection of the neurotransmitter glycine. Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords: pH Dependence Ionization Reduction potentials Structure–function relationships Glycine detection
1. Introduction Flavoproteins contain a versatile cofactor, which enables them to catalyze a wide variety of electron transfer processes of a single electron or of two electrons simultaneously. Glycine oxidase (GO, EC 1.4.3.19) is a member of the large dehydrogenase-oxidase class of flavoproteins [1–4]. The enzyme from Bacillus subtilis is a homotetrameric protein that contains 1 noncovalently bound FAD per 42 kDa of the protein monomer. It catalyzes the oxidative deamination of various, short chain D-amino acids and primary or secondary amines to yield the corresponding a-keto acids and hydrogen peroxide [1,2] as shown in (1): H H2-C NR2
COOH + O2 + H2O
C O
COOH + NHR2 + H2O2 (R = H, CH3)
(1)
Recently, a second GO from the thermophylic bacillus Geobacillus kaustophilus was isolated and characterized [5]. It is
Abbreviations: GO, Glycine oxidase (EC 1.4.3.19); DAAO, D-amino acid oxidase (EC 1.4.3.3); SOX, sarcosine oxidase (EC 1.5.3.1); LAAO, L-amino acid oxidase (EC 1.4.3.2). * Corresponding author. Tel.: þ332 421506; fax: þ332 421500. E-mail address: [email protected] (L. Pollegioni). 0300-9084/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2009.02.007
different from the B. subtilis GO in that it has a higher relative activity with D-alanine and D-proline and exhibits inhibition at high substrate concentrations. A large number of flavoproteins are known to oxidize amines and amino acids: these compounds play important metabolic roles that range from the utilization of carbon and/or nitrogen sources in bacteria to neurotransmission in mammalian brain. GO shares substrate specificity with D-amino acid oxidase (EC 1.4.3.3, DAAO) [6,7] and sarcosine oxidase (EC 1.5.3.1, SOX) [8–10], enzymes that are also of considerable interest for biotechnology. In particular, GO has recently acquired relevance in the investigation of neurotransmission mechanisms. By using it in cell cultures or tissue slices it can be verified whether the activation of NMDA-receptors is due to the coagonist glycine (which is a good substrate of GO) or to Dserine (which conversely is a good substrate of DAAO) [11]. Furthermore, the on-line detection of changes in the concentrations of these neurotransmitters is of paramount importance to investigate neurotransmission under physiological and pathological conditions, as well as to study the effect of new drugs against complex disorders such as schizophrenia and bipolar disease (see the recently developed DAAO-based microbiosensor [12]). This paper is a continuation of studies on GO [1,2,4,5,13] and we report on various properties of (recombinant) GO from B. subtilis, focusing on their pH dependence. These results, together with the available protein structure [14,15] and studies based on
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site-directed mutagenesis [4,16], promote two goals. On the one hand, they are expected to contribute to our knowledge of the structure–function relationships of GO and, specifically, to shed light on the factors that determine the specificity of GO compared to the other members of the family that use related substrates, such as monomeric SOX (MSOX), DAAO and L-amino acid oxidase. Secondly, the use of GO in assessing the role of glycine and D-serine in NMDA-receptor functionality requires a precise knowledge of its properties under a sufficiently broad range of conditions. 2. Materials and methods 2.1. pH Dependence experiments Recombinant B. subtilis GO was purified from Escherichia coli cells transformed with the pT7-(DBH)HisGO expression system, as previously described [1,2]: the recombinant enzyme contains an His-tag at the N-terminal end. Gel-permeation separations were performed on a Superdex 200 HR10/30 column using an Akta FPLC system (GE Healthcare). Kinetic experiments were carried out using the multicomponent buffer system containing 15 mM Tris, 15 mM phosphate, 15 mM sodium carbonate, 250 mM KCl, and 1% (v/v) glycerol; the KCl concentration being used to minimize the impact of ionic strength changes at different pH values. To avoid interference between the buffer components and the reducing system used to determine the reduction potentials, spectral experiments were performed using the following buffer system: in the 6.5–7.5 pH range, 50 mM potassium phosphate; at pH 8.0 and 8.5, 50 mM sodium pyrophosphate; in the 8.5–10.0 pH range, 25 mM sodium pyrophosphate and 25 mM carbonate buffer. All solutions contained 1% glycerol. 2.2. Activity assays GO Activity was determined at pH 8.5 and 25 C with 10 mM sarcosine as substrate by following the oxygen consumption [1,2]. One unit of GO is defined as the amount of enzyme that converts 1 mmol of substrate per minute at 25 C. Effects of pH in the range of 5–12 (Fig. 1A) were assessed using 10 mM sarcosine or glycine as substrate in the multicomponent buffer described above. For pH stability (Fig. 1B), the activity was assessed using the standard assay on enzyme samples (0.4 mg protein/mL) incubated for 60 min at 25 C and at various pH values. In order to calculate the steady-state kinetic parameters of the reaction at infinite (saturating) glycine and oxygen concentrations, the enzyme monitored turnover method was used in the pH range 6.5–9.5, as previously performed at pH 8.5 [13]. Data traces at 455 nm were analyzed by Kaleidagraph (Synergy Software, Reading, PA, USA). Steady state activity parameters were assessed based on the method of Gibson [17]. The pH dependence of data was fitted based on an equation for two dissociations (2):
Fig. 1. pH Dependence of activity parameters and of stability of GO. A) Activity with 10 mM glycine (B) or sarcosine (-), and B) stability of GO incubated for 60 min and then assayed at pH 8.5. Data are expressed as percentage of maximal enzyme activity. Bars indicate SE for three determinations. Lines through data points were obtained using eq. (2) and the following pKa values: pKa1 ¼ 7.6 and 8.0, pKa2 ¼ 11.7 and 10.8 for activity on glycine and sarcosine, respectively; pKa1 ¼ 6.1 and pKa2 ¼ 10.0 for stability. C) pH Dependence of steady-state coefficients of the reaction catalyzed by GO. The turnover parameters were assessed by the enzyme monitored turnover method [13,17] using varying concentrations of glycine as substrate (see Table 1). The lines through the data points were obtained as outlined above and reflect pK values z6.0 and 10.0.
where a is the limiting activity value at acidic pH, b is the calculated intermediate value, and c is the limiting activity value at basic pH.
adduct. Sulfite was added at 15 C in small volumes (1–10 ml) of concentrated stock solutions to GO (w10 mM) solutions in a suitable buffer at varying pH values [1]. Glycolate binding was analogously assessed using absorbance changes at w497 nm that accompany complex formation. Kd values were estimated based on the mass law. The pH dependence of Kd values was analyzed according to Dixon [18]; equation (3) describes the ionization of a group that combines with the ligand:
2.3. Ligand binding
i h pKd ¼ a log 1 þ 10pKa = 10pH
The dissociation constant for sulfite binding to GO was assessed spectrophotometrically by following changes in absorbance at 458 nm, which reflect the formation of the flavin N(5)-sulfite
where a is the pKd value at acidic pH. Equation (4) describes the ionization of a group that is altered following the formation of the enzyme-ligand complex:
Y ¼
i a þ b 10pHpKa1 = 1 þ 10pHpKa1 i h þ b c 10pHpKa2 = 1 þ 10pHpKa2 h
(2)
(3)
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i h pKd ¼ a log 1 þ 10pKa1 = 10pH i h þ log 1 þ 10pKa2 = 10pH
semiquinone form of GO reached during an experiment in the absence of the reference dye [21,22]:
ð4Þ
DEm ¼ 59 mV log K
(6)
K ¼ ½semiquinone2 =½reduced½oxidized
(7)
where a is the pKd value at acidic pH.
2.4. Studies of flavin semiquinone forms
3. Results
Solutions containing w8 mM GO in a suitable buffer at different pH values and containing 5 mM EDTA were made anaerobic in a closed, all-glass cuvette by repeated cycles of evacuation and flushing with O2-free argon. The enzyme was then photoreduced by light irradiation with a 250-W lamp at a distance of w20 cm in the presence of 0.5 mM 5-deazaflavin [19]. The progress of the photoreaction was followed spectrophotometrically. The extent of semiquinone formation was determined at 372 nm and the amount of reduced enzyme form was determined from absorbance changes at 508 nm, a wavelength corresponding to an isosbestic point between the oxidized/semiquinone flavin species. The thermodynamic stability of the semiquinone form was evaluated after incubation for 24 h at 15 C, following addition of 5 mM benzyl viologen to the enzyme solution; equilibration is generally attained at this point [1,2].
2.5. Determination of reduction potentials Xanthine, xanthine oxidase, and dyes were purchased from Sigma. The reduction potentials of GO were determined at 15 C by employing the dye-equilibration method [20] and using the xanthine/xanthine oxidase reduction system described by Massey [1,21]. The enzyme (w10 mM) was placed in an anaerobic cuvette in a buffer at varying pH values, containing 0.2 mM xanthine and w15 mM concentrations of the appropriate dye; to ensure rapid equilibration of reducing equivalents, 12.5 mM methyl viologen was also added. The cuvette was made anaerobic (see above) and the visible spectrum of the anaerobic enzyme was recorded. The reaction was started by adding xanthine oxidase (10–50 nM) from a side arm of the cuvette, and the absorbance spectra (w100) were collected until an increase was observed in the w600 nm peak, corresponding to reduction of the methyl viologen (this indicates completion of the enzyme reduction). The concentrations of the oxidized, semiquinone, and reduced forms of GO and of the oxidized and reduced forms of the reference dyes were determined from the absorbance values at appropriate wavelengths. The reduction potential, Eh, for the system at equilibrium was calculated from the Nernst equation (5):
Eh ¼ Em þ ð2:3RT=nFÞlogð½oxidized form=½reduced formÞ
(5)
where R ¼ the gas constant (8.31441 V K1 mol1); T ¼ the absolute temperature; F ¼ Faraday’s constant (9.6485381 104 C mol1); and n ¼ the number of electrochemical equivalents. All potential values are those versus the standard hydrogen electrode. The data were plotted as described previously [21]. The log([oxidized]/ [reduced]) couples for the enzyme is plotted against the log([oxidized]/[reduced]) concentration ratio for the dye; the slope of such plots is 1 when the two couples transfer the same number of electrons. The reduction potential for the couple of oxidized/semiquinone of GO (E1) was determined by plotting the ratio of their concentrations, and the potential for the couple semiquinone/ reduced (E2) was determined by plotting the ratio of the concentration of the semiquinone and reduced forms of the enzyme. The separation between the two single-electron reduction potentials is estimated from the maximal percentage of the
3.1. pH dependence of general properties Fig. 1A shows that the activity of GO increases with pH, reaches a maximum at about pH 9–10, and falls off at higher values when using sarcosine or glycine as substrates. The shape of the curve is narrower for sarcosine than for glycine but for both the maximal activity is observed at the same pH values (9.5–10.0). While the activity/pH profile of GO is similar to that observed for pig kidney DAAO, it differs from that found with yeast DAAOs, the latter having maxima in the pH range of 7.5–9.0 (depending on the enzyme source) [23]. The pH dependence of B. subtilis GO stability was assessed by measuring the activity after 60 min of incubation in the pH range 4–12; it is maximal at pH 7.0–8.0, showing a substantial decrease below pH 6.0 and above pH 9.5 (Fig. 1B), which is in good agreement with previous results [4]. Under similar conditions, GO from the thermophilus G. kaustophilus shows the highest stability at pH 7.0 but the decrease at acidic and basic pH values is less important [5]. Steady state data in the pH range 6–9.5 were obtained using the stopped-flow method described by Gibson [17]. The obtained parameters are listed in Table 1, and the pH profiles of kcat and of Km for glycine are depicted in Fig. 1C. kcat and Km for glycine show a pH dependence that is comparable to those reflected by the relative activity profiles (Fig. 1A) with maxima/minima at around pH 8.5– 9.0. On the contrary, for Km;O2 parameters no particular pH dependent trend is observable (Table 1). GO is a stable homotetramer whose oligomerization is not dependent on protein concentrations in the 0.01–13-mg/mL range [2]. There is no effect of pH on the GO oligomerization state, as shown using the gel-permeation chromatography: the elution volume is unchanged in the 6.5–10 pH range (not shown). 3.2. Semiquinone properties The stabilization of the red anionic semiquinone and the reactivity with sulfite are common features of flavoprotein oxidases [24]. In the pH range from 6.5 to 10.0, the irradiation of GO with visible light according to the method of [19] generates the red anionic flavin radical (see Fig. 1A in [1]). Formation of the latter is essentially 100% at pH < 8.5 and decreases to z90% at higher pH. The semiquinone is remarkably stable at neutral pH, >90% of the original amount being present upon incubation under anaerobic conditions at zpH 7.5. The rates of the decay increase substantially above and below this pH value (Fig. 2). The presence of benzyl viologen, a mediator of electron transfer, increases the rates of decay over the whole pH range yielding approx. twofold lower amount of semiquinone form. Based on these observations we assume that this radical species is stabilized both thermodynamically and kinetically, where both contributions appear to be pH dependent. 3.3. Sulfite and glycolate binding We have shown previously that sulfite forms a covalent adduct with the flavin cofactor N(5) position with a Kd in the mM range at
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Table 1 Steady-state kinetic parameters for the reaction of GO with glycine as a function of pH. pH
6.5 7.0 8.0 8.5 9.0 9.5
Steady state parameter (kcat ¼ 1/F0) (s1)
Fgly (M s) 103
FO2 (M s) 103
Fgly;O2 (M2 s) 106
Km,gly (mM)
Km;O2 (mM)
0.9 0.1 2.4 0.6 3.6 0.9 4.0 1.0 4.1 0.7 3.0 0.3
11.0 2.3 1.5 0.94 1.3 5.0
0.17 0.03 0.18 0.01 0.07 0.02 0.07 0.014 0.036 0.015 0.136 0.030
0.74 0.05 0.10 0.01 0.18 0.02 0.14 0.01 0.32 0.02 0.30 0.03
9.2 1.4 5.6 1.0 5.4 1.6 3.8 1.0 5.4 1.8 14.7 2.5
0.15 0.05 0.45 0.09 0.25 0.03 0.28 0.10 0.15 0.06 0.40 0.10
The data were obtained by the enzyme monitored turnover method [13] as described in Materials and Methods section.
neutral pH [1,25]. This is accompanied by disappearance of the oxidized flavin spectrum as observed during reduction with substrate. Kd values for sulfite binding were determined from the dependence of the absorbance at 458 nm from the added sulfite concentrations (not shown). Importantly, essentially complete formation of the complex is observed at all pH values tested. The Kd values increase exponentially with pH in the range 8–10 and reach a plateau at pH < 7 (Fig. 3 top). The data were analyzed according to the Dixon rules [18]. When this analysis is based on an equation reflecting a single ionization, the presence of a group with a pKa value of 8.05 0.14 can be inferred whereby its deprotonation inhibits sulfite binding (Fig. 3 top). A marginally better fit in an analogous analysis is obtained using a two-ionization equation that would reflect the presence of an ionizable group with a pKa value of 7.9 0.2 in the free enzyme that would shift to 10.1 0.3 in the complex. Unfortunately reliable data at pH > 10 could not be obtained due to the instability of the protein (see Fig. 1B). In analogy to the case for binding of glycolate (see below), and based on common assumptions we consider the presence of a second ionization with a pK z 10 as reasonably describing the situation, although the two types of analysis are equivalent. Previous, steady-state kinetic studies of GO using sarcosine as substrate indicated that glycolate is a competitive inhibitor (Ki ¼ 0.4 mM at pH 8.5) [1]. It binds to GO (Kd ¼ 0.6 mM, the tightest ligand for this flavoenzyme) altering the visible absorbance spectrum. This consists in the appearance of a shoulder at 500 nm and a shift of the 455 nm peak up to 460 nm [1]. We have investigated the pH dependence of Kd for glycolate binding. The results are
Fig. 2. pH Dependence of GO semiquinone formation and dismutation. (C): maximal relative amount of anionic semiquinone produced by irradiation as described in the Materials and Methods section and estimated based on its absorption spectrum [1]; (B): amount of semiquinone remaining after w15 h incubation in the dark at 15 C; (:): to the resulting species the redox mediator benzyl viologen was added anaerobically and the semiquinone was estimated after further 12 h of incubation in the dark.
depicted in Fig. 3 bottom based on the Dixon convention [18]. This infers the presence of a group on the free enzyme whose pKa value rises from 7.6 0.2 in the unliganded state to 9.2 0.2 upon glycolate binding. 3.4. Reduction potentials The absorbance spectra at different pH values and reduction states reported earlier [2] are a basis for interpreting the pH dependence of the redox potentials. The same holds also for the pH dependent spectral changes of the oxidized species that accompany
Fig. 3. pH Dependence of sulfite (top) and glycolate (bottom) binding to GO. The data are represented according to Dixon’s rules [18]. The fit was obtained using eq. (3) (curve - - - -, single ionization of a group that combines with the ligand) or based on eq. (4) (curves d, a group the ionization of which is affected by the binding). For sulfite binding the fit based on eq. (4) is marginally better compared to that based on eq. (3) (R ¼ 0.9834 vs. 0.9788); (for glycolate binding R ¼ 0.9910). The indicated pKa ¼ 7.95 for sulfite binding is the average of the pKa values obtained from the two fits (pKa 7.9 0.2 and pKa 8.05 0.14).
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ionization of the bound flavin N(3)–H (and that reflect a pKa ¼ 10.6) [2]. The reduction potentials for the two one-electron steps of flavin reduction in GO were thus determined spectroscopically by equilibrating the enzyme with an appropriate redox dye [1,21]. Anaerobic enzyme was first incubated in the presence of methyl viologen (Em ¼ 440 mV at pH 8.0) as the only dye. The spectral course of GO flavin reduction first displays the isosbestic points indicated in Fig. 4A corresponding to the conversion of the oxidized form into the semiquinone. Subsequently, the semiquinone is partially converted into the reduced species (Fig. 4B). The maximal amount of semiquinone formed during the reaction can be estimated from the absorbance changes at 372 and 458 nm, as shown in the inset to Fig. 4B: at pH 8.0 formation of this species approaches 100%, which is in agreement with the results obtained by photoirradiation (see Fig. 2). Since the (blue) neutral semiquinone is characterized by an absorption band centered in the 550–650 nm region [26] the absence of such an absorption (see Fig. 4) in the pH range down to 7 suggests that the pK of this species is <6. The semiquinone formation constant [21] and the separation between the potentials for single-electron transfer was estimated according to eqs. (6) and (7). The maximal amounts of semiquinone produced for GO and the DEm values are reported in Table 2. Subsequently, the same type of experiment was performed in the presence of a redox dye: representative results for determining E1 (oxidized-semiquinone couple) and E2 (semiquinone-reduced form couple) are shown in Fig. 5A and B. The E1 or the E2 potentials, respectively, can be estimated by secondary data evaluation such as the reported exemplarily in inset of Fig. 5, where the log([oxidized]/[semiquinone]) or the log([semiquinone]/[reduced]) of flavin species is plotted as a function of log([oxidized]/[reduced]) for the dye (gallocyanine or benzyl viologen) [20]. To study the effect of pH on the potentials of GO, the various dyes listed in Table 2 were used as redox indicators. The amount of the semiquinone species formed in these experiments is close to 100% at all pH values, which is in agreement with the large separation (DEm) between the single-electron reduction potentials E1 and E2 (Table 2). The E1 value shows essentially no pH dependence (Fig. 6): this is consistent with the first electron being transferred alone (i.e., without a simultaneous transfer of a proton) to yield an anionic semiquinone species, and with the pK of the semiquinone species being <7 (see above). On the other hand, a dependence on pH is evident for E2 and consequently also for the midpoint (Em) redox potentials (Fig. 6). It should be stated that the determination
of the E2 potentials at pH 7.5 is affected by the difficulty in measuring values lower than 400 mV with the experimental system employed. The slope of the pH dependence of Em is 23.4 mV/pH unit for GO, a value close to that of an overall twoelectrons/one proton transfer process. These observations indicate that a two single-electron transfer process ðE FADox þ 1e /E þ FAD seq and E FADseq þ 1e þ 1H /E FADred H Þ is favored for the free GO form. Under the same experimental conditions, the redox potentials at pH 7.5 were determined in the presence of 5 mM glycolate. In the absence of a dye as redox indicator, a lower amount of semiquinone is produced during the reduction (z70%) than with free GO, corresponding to a maximal separation between the single-electron transfer potentials of z150 mV (vs. 290 mV in the absence of the ligand). As reported in Table 2, and in comparison to the values determined for the free enzyme, the E2 value was z160 mV more positive. Glycolate binding to GO thus shifts the midpoint reduction potentials to more positive values by z80 mV and favors the two electrons transfer process. The addition of benzoate to DAAOs analogously decreases the stability of the semiquinone forms but modulates the redox properties differently. For example, a more negative Em is observed at pH 7.5 upon inhibitor binding (z50 mV in pig kidney DAAO and z10 mV in Rhodotorula gracilis DAAO) [27,28]. 4. Discussion 4.1. General properties We have analyzed in some detail the pH dependence of selected functional properties of B. subtilis GO. This enzyme shares some features with the members of the flavin dependent oxidase family, while it differs substantially in others. As opposed to e.g. DAAO it occurs in a stable homotetrameric state in a broad range of protein concentrations, a state that is not affected by pH in the 6.5–10 range either. The reasons for which GO is the only tetrameric member of the flavoprotein oxidase family acting on substrates carrying an amine functional group are still elusive. In the tetrameric state no restriction of the accessibility to the active site is apparent and an interaction between FAD molecules of different subunits is unlikely. Furthermore, an allosteric behavior did not emerge from the present or from previous investigations [13,14]. On the other hand it is worth noting that the physiological role of GO is to generate the glyoxylate imine that is required in the thiazole phosphate
Fig. 4. Anaerobic reduction of GO at pH 8.0. GO (12 mM) in 50 mM pyrophosphate buffer, pH 8.0, containing 10% glycerol, 250 mM xanthine, and 12.5 mM methyl viologen, was incubated anaerobically with 20 nM xanthine oxidase. (A) Spectra of GO before (thick line) and at 72, 102, 152, 212, 252, 302, 332, 392, and 452 min after the addition of xanthine oxidase. (B) Subsequent spectra at 632, 722, 812, 902, 992, 1082, 1172, 1262, and 1442 min. The arrows indicate isosbestic points (410 and 505 nm). Inset: estimation of the amount of produced semiquinone. The intersection of the lines corresponds to 100% formation.
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Table 2 Reduction potentials of Bacillus subtilis GO as function of pH. pH
GO Redox potentials (mV) Redox indicatora
E0m ðmVÞ
7.0
ITeS, ITS BV
24/60 359
DEm
E1
E2 323 5
300 (94%) 7.5
Ga/ITeS BV/Ph
179 6 39 9 [30 3]
þ3/43 359/239
354 5 [192 14] 300 (w100%) [147 (w70%)]
8.0
Ga/ITeS BV
197 7 [111 9] 38 8
28/59 359
367 8 300 (w100%)
8.5
Ga/ITeS BV
Em
35 7
203 8 40 3
53/74 359
392 10 300 (w100%)
216 7
The potentials were measured as described in the Materials and Methods section and using the indicated dyes as redox standards [20,21]. The values in brackets are those determined in the presence of 5 mM glycolate at pH 7.5. The DEm was estimated from the percentage of the semiquinone form (shown in parentheses) produced during reduction experiments performed in the presence of only methyl viologen and using eqs. (6) and (7). a ITeS, indigo tetrasulfonate; ITS: indigo trisulfonate; BV, benzyl viologen; Ga, gallocyanine; Ph, phenosafranine.
biosynthetic pathway [29]: such imines, however, do hydrolize readily in solvent, and it is thus conceivable that a transfer in an environment sealed from solvent is required. This lead to the hypothesis that the interaction of the tetramer with the partner enzyme(s) in the biosynthetic pathway is suited for the purpose (i.e., the thiazole synthase complexed with the sulfur carrier protein ThiS consists of a tetramer of ThiS/thiazole synthase heterodimers) [29]. GO shares with the flavoprotein oxidase family the stabilization of the (red) anionic semiquinone species. This species is stabilized mainly thermodynamically at neutral pH (z7.5), as is the case with yeast DAAO [27]. At higher and lower pH values, however, semiquinone stabilization has a kinetic component. The potential for the transfer of the first electron is mainly pH independent while the second is highly negative (E2 < 350 mV) and is pH dependent. The slope of the Em vs. pH shown in Fig. 6 is 24 mV/pH unit, which is consistent with the overall transfer of two electrons/one proton. This behavior is also compatible with the formation of the reduced flavin in its anionic form, as reported for DAAO [27,30] and L-amino acid oxidase [31]. The finding of a thermodynamic stabilization of the anionic semiquinone is seemingly in contrast to the presently accepted catalytic mechanism for the dehydrogenation of amino
acids by flavoproteins [32]: there is no experimental evidence for radical formation during catalysis and the binding of substrate might modulate the relative potentials for electron transfer. Thus, when a substrate analogue such as glycolate is bound the free energy of electron transfer is shifted in a direction that comparatively favors a two electron transfer process, this being reflected by a lower amount of semiquinone formed (70% vs. z100% in the presence/absence of 5 mM glycolate). This behavior parallels that observed with DAAOs using benzoate although there are substantial quantitative differences. For example, the amount of semiquinone formed at pH 7.5 is 70% in GO, 56% in Trigonopsis variabilis DAAO, and 20% in R. gracilis DAAO [27]. From this comparison it can be deduced that the presence of ligand(s) at the active site affects the free energy of electron transfer. This is, however, not the determinant for the mechanism of dehydrogenation. A peculiar characteristic of GO in the free form and at neutral pH is the more negative value of the midpoint potential for the twoelectron transfer (Em z 200 mV) as compared to DAAO (Em z 100 mV, specifically 108 in the mammalian and 91 mV in the yeast enzymes) [27,28] and to cholesterol oxidase (Em z 110 mV) [33] at pH 7.5. The significance of this finding is still elusive. A peculiarity of GO appears to be the decreasing ability of
Fig. 5. Estimation of the reduction potentials of GO at pH 8.0. (A) Selected spectra obtained during the course of the anaerobic reduction of GO for determination of E1. Conditions as for Fig. 4 with the addition of 12 mM gallocyanine as redox standard (Em ¼ 28 mV). Spectra were recorded before (thick line) and 50, 80, 120, 150, 200, 250, 360, 490, and 850 min after the addition of 20 nM xanthine oxidase. (B) Selected spectra obtained for determination of the potential for the second electron transfer (E2) recorded before (thick line) and 6, 10, 38, 50, 60, 92, 132, 252, 612, 852, and 1452 min after the addition of xanthine oxidase. Conditions as in panel A, however, with 16 mM benzyl viologen as redox standard (Em ¼ 359 mV). Insets: Nernst plots as described by [20,21]. The concentration of the oxidized and semiquinone forms of GO was estimated at 457 nm, after subtracting the dye’s contribution. The amount of the reduced enzyme was determined from the absorbance change at 508 nm, an isosbestic point for the oxidized/semiquinone forms (see Fig. 4A). The concentration of the oxidized and reduced forms of dyes was determined at 627 nm and at 602 nm for gallocyanine and benzyl viologen, respectively (a wavelength at which the contribution of the enzyme is negligible).
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Fig. 6. Effect of pH on the reduction potentials of GO, at 15 C. (C): E1, redox potential for the oxidized/semiquinone enzyme couple (slope ¼ 2.8); (-): E2, redox potential for the semiquinone/reduced enzyme couple (slope ¼ 45); (B): Em, redox potential for the oxidized/reduced enzyme couple (slope ¼ 24).
the protein to stabilize a negative charge at the flavin pyrimidine moiety with increasing pH and in comparison with e.g. DAAO. This is reflected by the lower stability of the semiquinone form at alkaline pH, the decrease of the affinity for sulfite with pH and the low Em. The pH dependence of Kd for sulfite binding (Fig. 3 top) identifies a group in the free enzyme with a pKa z 7.9 that is assumed to be increased to 10.1 upon formation of the adduct. Similarly, the pH dependence of glycolate binding (Fig. 3 bottom) indicates the presence of a group the pKa of which is shifted from z7.6 (free enzyme form) to z9.2 in the complex. This group is likely the same in the two cases and it thus appears reasonable to propose it to be Tyr246 in GO (Fig. 7). Its phenolic group is close to Arg302 and Arg329 where in particular the latter serves in fixation of the substrate carboxylate. In the absence of a negatively charged ligand the positive charge(s) of the mentioned arginine(s) are likely to lower the pKa of Tyr246 (from z9.2 in the presence of ligand) to the observed value of z7.6. The position of this group would be similar to that of Tyr223 in yeast DAAO. The latter group is the functionality with a pKa z 9.8 whose deprotonation weakens benzoate binding in DAAO (it forms a H–bond with one oxygen of the substrate carboxylate) and that was proposed to affect release of the imino acid product [34]. Major differences between GO and the ‘‘DAAO family’’ exist with respect to the stability and activity pH profiles. GO exhibits ‘‘bellshaped’’ behaviors, with maxima at pH 8–9.5 and regions of high instability at the pH extremes (Fig. 1). Yeast DAAO, in contrast, is stable at low pH and becomes gradually unstable at pH > 8, while pig kidney DAAO is moderately stable at low pH and becomes unstable at pH > 10, with a relative maximum around pH 9.5 [23]. Major differences between DAAO and GO are also apparent in the activity vs. pH profiles (Fig. 1): thus, while GO has rather narrow activity profiles with very low activity at pH < 7 and maxima at pH z 9.0, the yeast DAAOs have much broader profiles and mammalian DAAO does not reveal activity losses up to pH 10 [23]. For yeast DAAO the attribution of pK values derived from the pH
Fig. 7. Overlay of the active centers of GO, yeast DAAO and MSOX [14,15,32,37]. GO complexed with N-acetyl-glycine is in pink (NAcGly, PDB code: 1ng3), yeast DAAO in complex with D-trifluoroalanine is in green (CF3–D–Ala, PDB code: 1c0l), and MSOX in complex with dimethylglycine is in blue (diMeGly, PDB code: 1el5). Ligands are not shown. Oxygen atoms of side chains are in red, nitrogen atoms in blue and methionine sulfurs in yellow. The FAD molecules are colored in gold.
dependence of activity profiles has been discussed in detail previously [35]. In analogy to this, it can be assumed that the pK values at z6 and 10 reflect the ionization of the bound and free substrate amino group, although a contribution of enzyme functional groups has to be assumed. This conclusion is in line with that of Porter and Bright for the case of L-amino acid oxidase [36]. 4.2. Comparison of active center structure with that of related enzymes The chemistry underlying catalysis is probably very similar for the three flavoproteins GO, DAAO and MSOX. Accordingly, an inspection of the superpositions of the 3D-structures of these enzymes highlights a close positional correspondence of flavin, domains and side chains at the active center (Fig. 7). The major, notable difference is that in MSOX the substrate is bound in an orientation that is approximately a mirror image (in the flavin N(5)– N(10) axis) of that in the two other enzymes (Fig. 7) [14,15,32,37]. It should also be noted that with GO and DAAO the aC–H of an amino acid is transformed chemically, while in MSOX it is the C–H of an Nmethyl group. These two C–H functionalities differ to some extent in their reactivity; consequently they might require a specific tuning of the corresponding active sites for activation. The present structure will thus be the result of evolutionary pressure. From comparison of the active centers of GO, yeast DAAO and MSOX two groups of interactions can be distinguished. On the one hand there are those that affect the properties of the flavin, these concern mainly its pyrimidine moiety and are located around it. The second type regards substrate binding and its positioning and orientation. - Flavin pyrimidine ring interactions: in all these three enzymes there is a backbone region that encompasses the flavin
M. Pedotti et al. / Biochimie 91 (2009) 604–612
C(2)]O and N(3)–H functionalities with similar geometric and sterical features (Fig. 8). Thus N(3)–H forms tight H– bonds with backbone amide carbonyls (MSOX: to Ileu50 backbone, 2.98 Å; GO: to Met49, 2.97 Å; DAAO: to Asp54, backbone 2.97 Å). The distribution of backbone amide C]O and N–H functionalities is thus very similar in GO and yeast DAAO as shown in Fig. 8. Also, in both cases there are ahelices with their positive ends pointing towards the pyrimidine ring of the flavin. In MSOX the presence and position of these groups and helices is maintained, however, there is an additional Lys-3-NHþ 3 group (K348) at a distance of 2.78 Å of the pyrimidine C(2)]O. This likely is a factor in the better stabilization of a negative charge on the flavin in MSOX, and provides a rationale for the low pKa z 8.3 for the ionization of oxidized flavin N(3)–H [8]. This argument has been discussed by Schuman Jorns and collaborators [38] who have verified the concept nicely by implementing appropriate mutations in MSOX. By comparison the oxidized flavin N(3)–H pKa in GO and yeast DAAO is slightly increased from that of free flavin (pKa z 10) [39]. Similarly affected is the pKa of the flavin radical that is <6 in MSOX [9] and >9 in GO (Fig. 2). As expected, with MSOX the Arg49 to Lys mutation induces an increase of z100 mV of the potential for the first electron transfer [38]. This should be reflected also in the Em of the three proteins, as is in general the case for such situations in flavoproteins and as discussed by Efimov and McIntire [40]. Indeed the E1 for GO and yeast DAAO are 120 and 140 mV [27,28] lower compared to that of MSOX. A peculiarity of GO is the presence of a water molecule at 2.95 Å from the flavin C(2)]O that forms H-bridges to Met49-C]O backbone (3.2 Å, Fig. 8). A water located in a similar position is found in yeast DAAO, but is not observed in MSOX (Fig. 8 top).
611
- Substrate binding: in all cases, i.e., with DAAO, GO and MSOX the substrate-COO group is linked at the active site via a double bridge to an arginine (Figs. 7 and 8). With DAAO two Tyr–OH groups (Tyr223 at 2.75 Å and Tyr238 at 3.1 Å) fix one carboxylate, while in GO only one (Tyr246, 3.9 Å) interacts, although less tightly with the same group. A role comparable to that of the ‘‘second Tyr’’ in DAAO is probably exerted in GO by Arg329 that is located at 2.9 Å in a corresponding position. As already mentioned above, this set-up is different in MSOX where substrate binding is mirrored with respect to the flavin N(5)–N(10) axis (Fig. 8). Also in this case a biforked interaction exists with Arg52 (3.1 Å) and a second one at 2.8 Å with Lys348 that would correspond to those exerted by the ‘‘second Tyr’’ and by the arginine residue mentioned above. Intriguingly, His345, Arg52, and Lys348 in MSOX form a network with 2–3 positive charges that probably constitute an important factor in determining the chemical properties of the flavin. The present comparisons do not evidentiate basic differences in the ‘‘set-up’’ of the three enzyme active sites. On the contrary, the differences appear to be gradual, this suggesting basic mechanistic similarities for the dehydrogenation of the two types of C–H substrates. It also appears that in all three cases multiple interactions concur in fixing the substrate in a strictly conserved position in which the C–H function that expels the hydride during dehydrogenation is placed on top of the flavin position N(5) (Fig. 8, see also discussion in [32]). On the other hand, the tuning of the flavin reactivity, as manifested by the interactions with the pyrimidine moiety, appears to reflect the requirement of different flavin redox potentials for dehydrogenation of the two types of substrates, the aC–H vs. the N–CH3 groups. Finally, on comparing the three active site centers (Figs. 7 and 8) one previously unnoticed feature
Fig. 8. Orientation of ligands, charges and functional groups at the active centers of GO, yeast DAAO and MSOX (see Fig. 7) [14,15,32,37]. The top row highlights the similar modes of ligand binding and fixation and the positioning of methionines in the active site. The bottom row evidences the oxygen (red) or nitrogen (blue) atoms, belonging to amide functions that encompass the flavin pyrimidine ring (van der Waals representation); in the case of MSOX, also the nitrogen of K348 side chain is highlighted.
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emerges: in the ‘‘upper part’’ of the active sites there are methionine side chains (Met49 in GO, Met213 in yeast DAAO, Met245 in MSOX) that stem from different segments of the polypeptide main chains, but are located within a distance of z6 Å. While this is unlikely to be coincidental, a functional role was made evident only for yeast DAAO, since Met213 plays an important role in the selection of the substrate based on its side chain [41]. 5. Conclusions
[14]
[15]
[16]
[17] [18]
In conclusion, the discussed differences in properties highlight how the substrate specificity, the flavin reactivity, and kinetic properties have been finely tuned starting from similar protein scaffolds to acquire different physiological roles. This information is also expected to be useful for the biotechnological use of GO since it defines the possible framework for such applications. For example, human DAAO can be fully inhibited by benzoate at a concentration at which GO is not (the Kd values for benzoate binding are at least two orders of magnitude lower for the human enzyme than for GO at all pH values) [42]. This, in turn, might make it possible to develop a glycine-sensitive biosensor based on GO (similar to the one recently developed for D-serine using yeast DAAO) [12], and whose response is not modified by the use of the DAAO inhibitors employed as drugs to treat complex mental diseases such as schizophrenia.
[19]
[20]
[21] [22] [23]
[24] [25]
Acknowledgments
[26]
This work was supported by grants from Fondo di Ateneo per la Ricerca to L. Pollegioni. We thank the support from Consorzio Interuniversitario per le Biotecnologie (CIB). This paper is dedicated to the memory of Prof. Stefano Ferrari.
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Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
Catalytic and redox properties of glycine oxidase from Bacillus subtilis Mattia Pedotti, Sandro Ghisla, Laura Motteran, Gianluca Molla, Loredano Pollegioni* Department of Biotechnology and Molecular Sciences, University of Insubria, via J.H. Dunant 3, 21100 Varese, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 December 2008 Accepted 18 February 2009 Available online 28 February 2009
Glycine oxidase (GO) from Bacillus subtilis is a homotetrameric flavoprotein oxidase that catalyzes the oxidation of the amine functional group of sarcosine or glycine (and some D-amino acids) to yield the corresponding keto acids, ammonia/amine and H2O2. It shows optima at pH 7–8 for stability and pH 9–10 for activity, depending on the substrate. The tetrameric oligomeric state of the holoenzyme is not affected by pH in the 6.5–10 range. Free GO forms the anionic red semiquinone upon photoreduction. This species is thermodynamically stable, as indicated by the large separation of the two single-electron reduction potentials (DE 290 mV). The first potential is pH independent, while the second is dependent. The midpoint reduction potential exhibits a 23.4 mV/pH unit slope, which is consistent with an overall two-electrons/one-proton transfer in the reduction to yield anionic reduced flavin. In the presence of glycolate (a substrate analogue) and at pH 7.5 the potential for the semiquinone-reduced enzyme couple is shifted positively by w160 mV: this favors a two-electron transfer compared to the free enzyme. Binding of glycolate and sulfite is also affected by pH, showing dependencies that reflect the ionization of an active site residue with a pKa z 8.0. These results highlight substantial differences between GO and related flavoenzymes. This knowledge will facilitate biotechnological use of GO, e.g. as an innovative tool for the in vivo detection of the neurotransmitter glycine. Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords: pH Dependence Ionization Reduction potentials Structure–function relationships Glycine detection
1. Introduction Flavoproteins contain a versatile cofactor, which enables them to catalyze a wide variety of electron transfer processes of a single electron or of two electrons simultaneously. Glycine oxidase (GO, EC 1.4.3.19) is a member of the large dehydrogenase-oxidase class of flavoproteins [1–4]. The enzyme from Bacillus subtilis is a homotetrameric protein that contains 1 noncovalently bound FAD per 42 kDa of the protein monomer. It catalyzes the oxidative deamination of various, short chain D-amino acids and primary or secondary amines to yield the corresponding a-keto acids and hydrogen peroxide [1,2] as shown in (1): H H2-C NR2
COOH + O2 + H2O
C O
COOH + NHR2 + H2O2 (R = H, CH3)
(1)
Recently, a second GO from the thermophylic bacillus Geobacillus kaustophilus was isolated and characterized [5]. It is
Abbreviations: GO, Glycine oxidase (EC 1.4.3.19); DAAO, D-amino acid oxidase (EC 1.4.3.3); SOX, sarcosine oxidase (EC 1.5.3.1); LAAO, L-amino acid oxidase (EC 1.4.3.2). * Corresponding author. Tel.: þ332 421506; fax: þ332 421500. E-mail address: [email protected] (L. Pollegioni). 0300-9084/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2009.02.007
different from the B. subtilis GO in that it has a higher relative activity with D-alanine and D-proline and exhibits inhibition at high substrate concentrations. A large number of flavoproteins are known to oxidize amines and amino acids: these compounds play important metabolic roles that range from the utilization of carbon and/or nitrogen sources in bacteria to neurotransmission in mammalian brain. GO shares substrate specificity with D-amino acid oxidase (EC 1.4.3.3, DAAO) [6,7] and sarcosine oxidase (EC 1.5.3.1, SOX) [8–10], enzymes that are also of considerable interest for biotechnology. In particular, GO has recently acquired relevance in the investigation of neurotransmission mechanisms. By using it in cell cultures or tissue slices it can be verified whether the activation of NMDA-receptors is due to the coagonist glycine (which is a good substrate of GO) or to Dserine (which conversely is a good substrate of DAAO) [11]. Furthermore, the on-line detection of changes in the concentrations of these neurotransmitters is of paramount importance to investigate neurotransmission under physiological and pathological conditions, as well as to study the effect of new drugs against complex disorders such as schizophrenia and bipolar disease (see the recently developed DAAO-based microbiosensor [12]). This paper is a continuation of studies on GO [1,2,4,5,13] and we report on various properties of (recombinant) GO from B. subtilis, focusing on their pH dependence. These results, together with the available protein structure [14,15] and studies based on
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605
site-directed mutagenesis [4,16], promote two goals. On the one hand, they are expected to contribute to our knowledge of the structure–function relationships of GO and, specifically, to shed light on the factors that determine the specificity of GO compared to the other members of the family that use related substrates, such as monomeric SOX (MSOX), DAAO and L-amino acid oxidase. Secondly, the use of GO in assessing the role of glycine and D-serine in NMDA-receptor functionality requires a precise knowledge of its properties under a sufficiently broad range of conditions. 2. Materials and methods 2.1. pH Dependence experiments Recombinant B. subtilis GO was purified from Escherichia coli cells transformed with the pT7-(DBH)HisGO expression system, as previously described [1,2]: the recombinant enzyme contains an His-tag at the N-terminal end. Gel-permeation separations were performed on a Superdex 200 HR10/30 column using an Akta FPLC system (GE Healthcare). Kinetic experiments were carried out using the multicomponent buffer system containing 15 mM Tris, 15 mM phosphate, 15 mM sodium carbonate, 250 mM KCl, and 1% (v/v) glycerol; the KCl concentration being used to minimize the impact of ionic strength changes at different pH values. To avoid interference between the buffer components and the reducing system used to determine the reduction potentials, spectral experiments were performed using the following buffer system: in the 6.5–7.5 pH range, 50 mM potassium phosphate; at pH 8.0 and 8.5, 50 mM sodium pyrophosphate; in the 8.5–10.0 pH range, 25 mM sodium pyrophosphate and 25 mM carbonate buffer. All solutions contained 1% glycerol. 2.2. Activity assays GO Activity was determined at pH 8.5 and 25 C with 10 mM sarcosine as substrate by following the oxygen consumption [1,2]. One unit of GO is defined as the amount of enzyme that converts 1 mmol of substrate per minute at 25 C. Effects of pH in the range of 5–12 (Fig. 1A) were assessed using 10 mM sarcosine or glycine as substrate in the multicomponent buffer described above. For pH stability (Fig. 1B), the activity was assessed using the standard assay on enzyme samples (0.4 mg protein/mL) incubated for 60 min at 25 C and at various pH values. In order to calculate the steady-state kinetic parameters of the reaction at infinite (saturating) glycine and oxygen concentrations, the enzyme monitored turnover method was used in the pH range 6.5–9.5, as previously performed at pH 8.5 [13]. Data traces at 455 nm were analyzed by Kaleidagraph (Synergy Software, Reading, PA, USA). Steady state activity parameters were assessed based on the method of Gibson [17]. The pH dependence of data was fitted based on an equation for two dissociations (2):
Fig. 1. pH Dependence of activity parameters and of stability of GO. A) Activity with 10 mM glycine (B) or sarcosine (-), and B) stability of GO incubated for 60 min and then assayed at pH 8.5. Data are expressed as percentage of maximal enzyme activity. Bars indicate SE for three determinations. Lines through data points were obtained using eq. (2) and the following pKa values: pKa1 ¼ 7.6 and 8.0, pKa2 ¼ 11.7 and 10.8 for activity on glycine and sarcosine, respectively; pKa1 ¼ 6.1 and pKa2 ¼ 10.0 for stability. C) pH Dependence of steady-state coefficients of the reaction catalyzed by GO. The turnover parameters were assessed by the enzyme monitored turnover method [13,17] using varying concentrations of glycine as substrate (see Table 1). The lines through the data points were obtained as outlined above and reflect pK values z6.0 and 10.0.
where a is the limiting activity value at acidic pH, b is the calculated intermediate value, and c is the limiting activity value at basic pH.
adduct. Sulfite was added at 15 C in small volumes (1–10 ml) of concentrated stock solutions to GO (w10 mM) solutions in a suitable buffer at varying pH values [1]. Glycolate binding was analogously assessed using absorbance changes at w497 nm that accompany complex formation. Kd values were estimated based on the mass law. The pH dependence of Kd values was analyzed according to Dixon [18]; equation (3) describes the ionization of a group that combines with the ligand:
2.3. Ligand binding
i h pKd ¼ a log 1 þ 10pKa = 10pH
The dissociation constant for sulfite binding to GO was assessed spectrophotometrically by following changes in absorbance at 458 nm, which reflect the formation of the flavin N(5)-sulfite
where a is the pKd value at acidic pH. Equation (4) describes the ionization of a group that is altered following the formation of the enzyme-ligand complex:
Y ¼
i a þ b 10pHpKa1 = 1 þ 10pHpKa1 i h þ b c 10pHpKa2 = 1 þ 10pHpKa2 h
(2)
(3)
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i h pKd ¼ a log 1 þ 10pKa1 = 10pH i h þ log 1 þ 10pKa2 = 10pH
semiquinone form of GO reached during an experiment in the absence of the reference dye [21,22]:
ð4Þ
DEm ¼ 59 mV log K
(6)
K ¼ ½semiquinone2 =½reduced½oxidized
(7)
where a is the pKd value at acidic pH.
2.4. Studies of flavin semiquinone forms
3. Results
Solutions containing w8 mM GO in a suitable buffer at different pH values and containing 5 mM EDTA were made anaerobic in a closed, all-glass cuvette by repeated cycles of evacuation and flushing with O2-free argon. The enzyme was then photoreduced by light irradiation with a 250-W lamp at a distance of w20 cm in the presence of 0.5 mM 5-deazaflavin [19]. The progress of the photoreaction was followed spectrophotometrically. The extent of semiquinone formation was determined at 372 nm and the amount of reduced enzyme form was determined from absorbance changes at 508 nm, a wavelength corresponding to an isosbestic point between the oxidized/semiquinone flavin species. The thermodynamic stability of the semiquinone form was evaluated after incubation for 24 h at 15 C, following addition of 5 mM benzyl viologen to the enzyme solution; equilibration is generally attained at this point [1,2].
2.5. Determination of reduction potentials Xanthine, xanthine oxidase, and dyes were purchased from Sigma. The reduction potentials of GO were determined at 15 C by employing the dye-equilibration method [20] and using the xanthine/xanthine oxidase reduction system described by Massey [1,21]. The enzyme (w10 mM) was placed in an anaerobic cuvette in a buffer at varying pH values, containing 0.2 mM xanthine and w15 mM concentrations of the appropriate dye; to ensure rapid equilibration of reducing equivalents, 12.5 mM methyl viologen was also added. The cuvette was made anaerobic (see above) and the visible spectrum of the anaerobic enzyme was recorded. The reaction was started by adding xanthine oxidase (10–50 nM) from a side arm of the cuvette, and the absorbance spectra (w100) were collected until an increase was observed in the w600 nm peak, corresponding to reduction of the methyl viologen (this indicates completion of the enzyme reduction). The concentrations of the oxidized, semiquinone, and reduced forms of GO and of the oxidized and reduced forms of the reference dyes were determined from the absorbance values at appropriate wavelengths. The reduction potential, Eh, for the system at equilibrium was calculated from the Nernst equation (5):
Eh ¼ Em þ ð2:3RT=nFÞlogð½oxidized form=½reduced formÞ
(5)
where R ¼ the gas constant (8.31441 V K1 mol1); T ¼ the absolute temperature; F ¼ Faraday’s constant (9.6485381 104 C mol1); and n ¼ the number of electrochemical equivalents. All potential values are those versus the standard hydrogen electrode. The data were plotted as described previously [21]. The log([oxidized]/ [reduced]) couples for the enzyme is plotted against the log([oxidized]/[reduced]) concentration ratio for the dye; the slope of such plots is 1 when the two couples transfer the same number of electrons. The reduction potential for the couple of oxidized/semiquinone of GO (E1) was determined by plotting the ratio of their concentrations, and the potential for the couple semiquinone/ reduced (E2) was determined by plotting the ratio of the concentration of the semiquinone and reduced forms of the enzyme. The separation between the two single-electron reduction potentials is estimated from the maximal percentage of the
3.1. pH dependence of general properties Fig. 1A shows that the activity of GO increases with pH, reaches a maximum at about pH 9–10, and falls off at higher values when using sarcosine or glycine as substrates. The shape of the curve is narrower for sarcosine than for glycine but for both the maximal activity is observed at the same pH values (9.5–10.0). While the activity/pH profile of GO is similar to that observed for pig kidney DAAO, it differs from that found with yeast DAAOs, the latter having maxima in the pH range of 7.5–9.0 (depending on the enzyme source) [23]. The pH dependence of B. subtilis GO stability was assessed by measuring the activity after 60 min of incubation in the pH range 4–12; it is maximal at pH 7.0–8.0, showing a substantial decrease below pH 6.0 and above pH 9.5 (Fig. 1B), which is in good agreement with previous results [4]. Under similar conditions, GO from the thermophilus G. kaustophilus shows the highest stability at pH 7.0 but the decrease at acidic and basic pH values is less important [5]. Steady state data in the pH range 6–9.5 were obtained using the stopped-flow method described by Gibson [17]. The obtained parameters are listed in Table 1, and the pH profiles of kcat and of Km for glycine are depicted in Fig. 1C. kcat and Km for glycine show a pH dependence that is comparable to those reflected by the relative activity profiles (Fig. 1A) with maxima/minima at around pH 8.5– 9.0. On the contrary, for Km;O2 parameters no particular pH dependent trend is observable (Table 1). GO is a stable homotetramer whose oligomerization is not dependent on protein concentrations in the 0.01–13-mg/mL range [2]. There is no effect of pH on the GO oligomerization state, as shown using the gel-permeation chromatography: the elution volume is unchanged in the 6.5–10 pH range (not shown). 3.2. Semiquinone properties The stabilization of the red anionic semiquinone and the reactivity with sulfite are common features of flavoprotein oxidases [24]. In the pH range from 6.5 to 10.0, the irradiation of GO with visible light according to the method of [19] generates the red anionic flavin radical (see Fig. 1A in [1]). Formation of the latter is essentially 100% at pH < 8.5 and decreases to z90% at higher pH. The semiquinone is remarkably stable at neutral pH, >90% of the original amount being present upon incubation under anaerobic conditions at zpH 7.5. The rates of the decay increase substantially above and below this pH value (Fig. 2). The presence of benzyl viologen, a mediator of electron transfer, increases the rates of decay over the whole pH range yielding approx. twofold lower amount of semiquinone form. Based on these observations we assume that this radical species is stabilized both thermodynamically and kinetically, where both contributions appear to be pH dependent. 3.3. Sulfite and glycolate binding We have shown previously that sulfite forms a covalent adduct with the flavin cofactor N(5) position with a Kd in the mM range at
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Table 1 Steady-state kinetic parameters for the reaction of GO with glycine as a function of pH. pH
6.5 7.0 8.0 8.5 9.0 9.5
Steady state parameter (kcat ¼ 1/F0) (s1)
Fgly (M s) 103
FO2 (M s) 103
Fgly;O2 (M2 s) 106
Km,gly (mM)
Km;O2 (mM)
0.9 0.1 2.4 0.6 3.6 0.9 4.0 1.0 4.1 0.7 3.0 0.3
11.0 2.3 1.5 0.94 1.3 5.0
0.17 0.03 0.18 0.01 0.07 0.02 0.07 0.014 0.036 0.015 0.136 0.030
0.74 0.05 0.10 0.01 0.18 0.02 0.14 0.01 0.32 0.02 0.30 0.03
9.2 1.4 5.6 1.0 5.4 1.6 3.8 1.0 5.4 1.8 14.7 2.5
0.15 0.05 0.45 0.09 0.25 0.03 0.28 0.10 0.15 0.06 0.40 0.10
The data were obtained by the enzyme monitored turnover method [13] as described in Materials and Methods section.
neutral pH [1,25]. This is accompanied by disappearance of the oxidized flavin spectrum as observed during reduction with substrate. Kd values for sulfite binding were determined from the dependence of the absorbance at 458 nm from the added sulfite concentrations (not shown). Importantly, essentially complete formation of the complex is observed at all pH values tested. The Kd values increase exponentially with pH in the range 8–10 and reach a plateau at pH < 7 (Fig. 3 top). The data were analyzed according to the Dixon rules [18]. When this analysis is based on an equation reflecting a single ionization, the presence of a group with a pKa value of 8.05 0.14 can be inferred whereby its deprotonation inhibits sulfite binding (Fig. 3 top). A marginally better fit in an analogous analysis is obtained using a two-ionization equation that would reflect the presence of an ionizable group with a pKa value of 7.9 0.2 in the free enzyme that would shift to 10.1 0.3 in the complex. Unfortunately reliable data at pH > 10 could not be obtained due to the instability of the protein (see Fig. 1B). In analogy to the case for binding of glycolate (see below), and based on common assumptions we consider the presence of a second ionization with a pK z 10 as reasonably describing the situation, although the two types of analysis are equivalent. Previous, steady-state kinetic studies of GO using sarcosine as substrate indicated that glycolate is a competitive inhibitor (Ki ¼ 0.4 mM at pH 8.5) [1]. It binds to GO (Kd ¼ 0.6 mM, the tightest ligand for this flavoenzyme) altering the visible absorbance spectrum. This consists in the appearance of a shoulder at 500 nm and a shift of the 455 nm peak up to 460 nm [1]. We have investigated the pH dependence of Kd for glycolate binding. The results are
Fig. 2. pH Dependence of GO semiquinone formation and dismutation. (C): maximal relative amount of anionic semiquinone produced by irradiation as described in the Materials and Methods section and estimated based on its absorption spectrum [1]; (B): amount of semiquinone remaining after w15 h incubation in the dark at 15 C; (:): to the resulting species the redox mediator benzyl viologen was added anaerobically and the semiquinone was estimated after further 12 h of incubation in the dark.
depicted in Fig. 3 bottom based on the Dixon convention [18]. This infers the presence of a group on the free enzyme whose pKa value rises from 7.6 0.2 in the unliganded state to 9.2 0.2 upon glycolate binding. 3.4. Reduction potentials The absorbance spectra at different pH values and reduction states reported earlier [2] are a basis for interpreting the pH dependence of the redox potentials. The same holds also for the pH dependent spectral changes of the oxidized species that accompany
Fig. 3. pH Dependence of sulfite (top) and glycolate (bottom) binding to GO. The data are represented according to Dixon’s rules [18]. The fit was obtained using eq. (3) (curve - - - -, single ionization of a group that combines with the ligand) or based on eq. (4) (curves d, a group the ionization of which is affected by the binding). For sulfite binding the fit based on eq. (4) is marginally better compared to that based on eq. (3) (R ¼ 0.9834 vs. 0.9788); (for glycolate binding R ¼ 0.9910). The indicated pKa ¼ 7.95 for sulfite binding is the average of the pKa values obtained from the two fits (pKa 7.9 0.2 and pKa 8.05 0.14).
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ionization of the bound flavin N(3)–H (and that reflect a pKa ¼ 10.6) [2]. The reduction potentials for the two one-electron steps of flavin reduction in GO were thus determined spectroscopically by equilibrating the enzyme with an appropriate redox dye [1,21]. Anaerobic enzyme was first incubated in the presence of methyl viologen (Em ¼ 440 mV at pH 8.0) as the only dye. The spectral course of GO flavin reduction first displays the isosbestic points indicated in Fig. 4A corresponding to the conversion of the oxidized form into the semiquinone. Subsequently, the semiquinone is partially converted into the reduced species (Fig. 4B). The maximal amount of semiquinone formed during the reaction can be estimated from the absorbance changes at 372 and 458 nm, as shown in the inset to Fig. 4B: at pH 8.0 formation of this species approaches 100%, which is in agreement with the results obtained by photoirradiation (see Fig. 2). Since the (blue) neutral semiquinone is characterized by an absorption band centered in the 550–650 nm region [26] the absence of such an absorption (see Fig. 4) in the pH range down to 7 suggests that the pK of this species is <6. The semiquinone formation constant [21] and the separation between the potentials for single-electron transfer was estimated according to eqs. (6) and (7). The maximal amounts of semiquinone produced for GO and the DEm values are reported in Table 2. Subsequently, the same type of experiment was performed in the presence of a redox dye: representative results for determining E1 (oxidized-semiquinone couple) and E2 (semiquinone-reduced form couple) are shown in Fig. 5A and B. The E1 or the E2 potentials, respectively, can be estimated by secondary data evaluation such as the reported exemplarily in inset of Fig. 5, where the log([oxidized]/[semiquinone]) or the log([semiquinone]/[reduced]) of flavin species is plotted as a function of log([oxidized]/[reduced]) for the dye (gallocyanine or benzyl viologen) [20]. To study the effect of pH on the potentials of GO, the various dyes listed in Table 2 were used as redox indicators. The amount of the semiquinone species formed in these experiments is close to 100% at all pH values, which is in agreement with the large separation (DEm) between the single-electron reduction potentials E1 and E2 (Table 2). The E1 value shows essentially no pH dependence (Fig. 6): this is consistent with the first electron being transferred alone (i.e., without a simultaneous transfer of a proton) to yield an anionic semiquinone species, and with the pK of the semiquinone species being <7 (see above). On the other hand, a dependence on pH is evident for E2 and consequently also for the midpoint (Em) redox potentials (Fig. 6). It should be stated that the determination
of the E2 potentials at pH 7.5 is affected by the difficulty in measuring values lower than 400 mV with the experimental system employed. The slope of the pH dependence of Em is 23.4 mV/pH unit for GO, a value close to that of an overall twoelectrons/one proton transfer process. These observations indicate that a two single-electron transfer process ðE FADox þ 1e /E þ FAD seq and E FADseq þ 1e þ 1H /E FADred H Þ is favored for the free GO form. Under the same experimental conditions, the redox potentials at pH 7.5 were determined in the presence of 5 mM glycolate. In the absence of a dye as redox indicator, a lower amount of semiquinone is produced during the reduction (z70%) than with free GO, corresponding to a maximal separation between the single-electron transfer potentials of z150 mV (vs. 290 mV in the absence of the ligand). As reported in Table 2, and in comparison to the values determined for the free enzyme, the E2 value was z160 mV more positive. Glycolate binding to GO thus shifts the midpoint reduction potentials to more positive values by z80 mV and favors the two electrons transfer process. The addition of benzoate to DAAOs analogously decreases the stability of the semiquinone forms but modulates the redox properties differently. For example, a more negative Em is observed at pH 7.5 upon inhibitor binding (z50 mV in pig kidney DAAO and z10 mV in Rhodotorula gracilis DAAO) [27,28]. 4. Discussion 4.1. General properties We have analyzed in some detail the pH dependence of selected functional properties of B. subtilis GO. This enzyme shares some features with the members of the flavin dependent oxidase family, while it differs substantially in others. As opposed to e.g. DAAO it occurs in a stable homotetrameric state in a broad range of protein concentrations, a state that is not affected by pH in the 6.5–10 range either. The reasons for which GO is the only tetrameric member of the flavoprotein oxidase family acting on substrates carrying an amine functional group are still elusive. In the tetrameric state no restriction of the accessibility to the active site is apparent and an interaction between FAD molecules of different subunits is unlikely. Furthermore, an allosteric behavior did not emerge from the present or from previous investigations [13,14]. On the other hand it is worth noting that the physiological role of GO is to generate the glyoxylate imine that is required in the thiazole phosphate
Fig. 4. Anaerobic reduction of GO at pH 8.0. GO (12 mM) in 50 mM pyrophosphate buffer, pH 8.0, containing 10% glycerol, 250 mM xanthine, and 12.5 mM methyl viologen, was incubated anaerobically with 20 nM xanthine oxidase. (A) Spectra of GO before (thick line) and at 72, 102, 152, 212, 252, 302, 332, 392, and 452 min after the addition of xanthine oxidase. (B) Subsequent spectra at 632, 722, 812, 902, 992, 1082, 1172, 1262, and 1442 min. The arrows indicate isosbestic points (410 and 505 nm). Inset: estimation of the amount of produced semiquinone. The intersection of the lines corresponds to 100% formation.
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Table 2 Reduction potentials of Bacillus subtilis GO as function of pH. pH
GO Redox potentials (mV) Redox indicatora
E0m ðmVÞ
7.0
ITeS, ITS BV
24/60 359
DEm
E1
E2 323 5
300 (94%) 7.5
Ga/ITeS BV/Ph
179 6 39 9 [30 3]
þ3/43 359/239
354 5 [192 14] 300 (w100%) [147 (w70%)]
8.0
Ga/ITeS BV
197 7 [111 9] 38 8
28/59 359
367 8 300 (w100%)
8.5
Ga/ITeS BV
Em
35 7
203 8 40 3
53/74 359
392 10 300 (w100%)
216 7
The potentials were measured as described in the Materials and Methods section and using the indicated dyes as redox standards [20,21]. The values in brackets are those determined in the presence of 5 mM glycolate at pH 7.5. The DEm was estimated from the percentage of the semiquinone form (shown in parentheses) produced during reduction experiments performed in the presence of only methyl viologen and using eqs. (6) and (7). a ITeS, indigo tetrasulfonate; ITS: indigo trisulfonate; BV, benzyl viologen; Ga, gallocyanine; Ph, phenosafranine.
biosynthetic pathway [29]: such imines, however, do hydrolize readily in solvent, and it is thus conceivable that a transfer in an environment sealed from solvent is required. This lead to the hypothesis that the interaction of the tetramer with the partner enzyme(s) in the biosynthetic pathway is suited for the purpose (i.e., the thiazole synthase complexed with the sulfur carrier protein ThiS consists of a tetramer of ThiS/thiazole synthase heterodimers) [29]. GO shares with the flavoprotein oxidase family the stabilization of the (red) anionic semiquinone species. This species is stabilized mainly thermodynamically at neutral pH (z7.5), as is the case with yeast DAAO [27]. At higher and lower pH values, however, semiquinone stabilization has a kinetic component. The potential for the transfer of the first electron is mainly pH independent while the second is highly negative (E2 < 350 mV) and is pH dependent. The slope of the Em vs. pH shown in Fig. 6 is 24 mV/pH unit, which is consistent with the overall transfer of two electrons/one proton. This behavior is also compatible with the formation of the reduced flavin in its anionic form, as reported for DAAO [27,30] and L-amino acid oxidase [31]. The finding of a thermodynamic stabilization of the anionic semiquinone is seemingly in contrast to the presently accepted catalytic mechanism for the dehydrogenation of amino
acids by flavoproteins [32]: there is no experimental evidence for radical formation during catalysis and the binding of substrate might modulate the relative potentials for electron transfer. Thus, when a substrate analogue such as glycolate is bound the free energy of electron transfer is shifted in a direction that comparatively favors a two electron transfer process, this being reflected by a lower amount of semiquinone formed (70% vs. z100% in the presence/absence of 5 mM glycolate). This behavior parallels that observed with DAAOs using benzoate although there are substantial quantitative differences. For example, the amount of semiquinone formed at pH 7.5 is 70% in GO, 56% in Trigonopsis variabilis DAAO, and 20% in R. gracilis DAAO [27]. From this comparison it can be deduced that the presence of ligand(s) at the active site affects the free energy of electron transfer. This is, however, not the determinant for the mechanism of dehydrogenation. A peculiar characteristic of GO in the free form and at neutral pH is the more negative value of the midpoint potential for the twoelectron transfer (Em z 200 mV) as compared to DAAO (Em z 100 mV, specifically 108 in the mammalian and 91 mV in the yeast enzymes) [27,28] and to cholesterol oxidase (Em z 110 mV) [33] at pH 7.5. The significance of this finding is still elusive. A peculiarity of GO appears to be the decreasing ability of
Fig. 5. Estimation of the reduction potentials of GO at pH 8.0. (A) Selected spectra obtained during the course of the anaerobic reduction of GO for determination of E1. Conditions as for Fig. 4 with the addition of 12 mM gallocyanine as redox standard (Em ¼ 28 mV). Spectra were recorded before (thick line) and 50, 80, 120, 150, 200, 250, 360, 490, and 850 min after the addition of 20 nM xanthine oxidase. (B) Selected spectra obtained for determination of the potential for the second electron transfer (E2) recorded before (thick line) and 6, 10, 38, 50, 60, 92, 132, 252, 612, 852, and 1452 min after the addition of xanthine oxidase. Conditions as in panel A, however, with 16 mM benzyl viologen as redox standard (Em ¼ 359 mV). Insets: Nernst plots as described by [20,21]. The concentration of the oxidized and semiquinone forms of GO was estimated at 457 nm, after subtracting the dye’s contribution. The amount of the reduced enzyme was determined from the absorbance change at 508 nm, an isosbestic point for the oxidized/semiquinone forms (see Fig. 4A). The concentration of the oxidized and reduced forms of dyes was determined at 627 nm and at 602 nm for gallocyanine and benzyl viologen, respectively (a wavelength at which the contribution of the enzyme is negligible).
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Fig. 6. Effect of pH on the reduction potentials of GO, at 15 C. (C): E1, redox potential for the oxidized/semiquinone enzyme couple (slope ¼ 2.8); (-): E2, redox potential for the semiquinone/reduced enzyme couple (slope ¼ 45); (B): Em, redox potential for the oxidized/reduced enzyme couple (slope ¼ 24).
the protein to stabilize a negative charge at the flavin pyrimidine moiety with increasing pH and in comparison with e.g. DAAO. This is reflected by the lower stability of the semiquinone form at alkaline pH, the decrease of the affinity for sulfite with pH and the low Em. The pH dependence of Kd for sulfite binding (Fig. 3 top) identifies a group in the free enzyme with a pKa z 7.9 that is assumed to be increased to 10.1 upon formation of the adduct. Similarly, the pH dependence of glycolate binding (Fig. 3 bottom) indicates the presence of a group the pKa of which is shifted from z7.6 (free enzyme form) to z9.2 in the complex. This group is likely the same in the two cases and it thus appears reasonable to propose it to be Tyr246 in GO (Fig. 7). Its phenolic group is close to Arg302 and Arg329 where in particular the latter serves in fixation of the substrate carboxylate. In the absence of a negatively charged ligand the positive charge(s) of the mentioned arginine(s) are likely to lower the pKa of Tyr246 (from z9.2 in the presence of ligand) to the observed value of z7.6. The position of this group would be similar to that of Tyr223 in yeast DAAO. The latter group is the functionality with a pKa z 9.8 whose deprotonation weakens benzoate binding in DAAO (it forms a H–bond with one oxygen of the substrate carboxylate) and that was proposed to affect release of the imino acid product [34]. Major differences between GO and the ‘‘DAAO family’’ exist with respect to the stability and activity pH profiles. GO exhibits ‘‘bellshaped’’ behaviors, with maxima at pH 8–9.5 and regions of high instability at the pH extremes (Fig. 1). Yeast DAAO, in contrast, is stable at low pH and becomes gradually unstable at pH > 8, while pig kidney DAAO is moderately stable at low pH and becomes unstable at pH > 10, with a relative maximum around pH 9.5 [23]. Major differences between DAAO and GO are also apparent in the activity vs. pH profiles (Fig. 1): thus, while GO has rather narrow activity profiles with very low activity at pH < 7 and maxima at pH z 9.0, the yeast DAAOs have much broader profiles and mammalian DAAO does not reveal activity losses up to pH 10 [23]. For yeast DAAO the attribution of pK values derived from the pH
Fig. 7. Overlay of the active centers of GO, yeast DAAO and MSOX [14,15,32,37]. GO complexed with N-acetyl-glycine is in pink (NAcGly, PDB code: 1ng3), yeast DAAO in complex with D-trifluoroalanine is in green (CF3–D–Ala, PDB code: 1c0l), and MSOX in complex with dimethylglycine is in blue (diMeGly, PDB code: 1el5). Ligands are not shown. Oxygen atoms of side chains are in red, nitrogen atoms in blue and methionine sulfurs in yellow. The FAD molecules are colored in gold.
dependence of activity profiles has been discussed in detail previously [35]. In analogy to this, it can be assumed that the pK values at z6 and 10 reflect the ionization of the bound and free substrate amino group, although a contribution of enzyme functional groups has to be assumed. This conclusion is in line with that of Porter and Bright for the case of L-amino acid oxidase [36]. 4.2. Comparison of active center structure with that of related enzymes The chemistry underlying catalysis is probably very similar for the three flavoproteins GO, DAAO and MSOX. Accordingly, an inspection of the superpositions of the 3D-structures of these enzymes highlights a close positional correspondence of flavin, domains and side chains at the active center (Fig. 7). The major, notable difference is that in MSOX the substrate is bound in an orientation that is approximately a mirror image (in the flavin N(5)– N(10) axis) of that in the two other enzymes (Fig. 7) [14,15,32,37]. It should also be noted that with GO and DAAO the aC–H of an amino acid is transformed chemically, while in MSOX it is the C–H of an Nmethyl group. These two C–H functionalities differ to some extent in their reactivity; consequently they might require a specific tuning of the corresponding active sites for activation. The present structure will thus be the result of evolutionary pressure. From comparison of the active centers of GO, yeast DAAO and MSOX two groups of interactions can be distinguished. On the one hand there are those that affect the properties of the flavin, these concern mainly its pyrimidine moiety and are located around it. The second type regards substrate binding and its positioning and orientation. - Flavin pyrimidine ring interactions: in all these three enzymes there is a backbone region that encompasses the flavin
M. Pedotti et al. / Biochimie 91 (2009) 604–612
C(2)]O and N(3)–H functionalities with similar geometric and sterical features (Fig. 8). Thus N(3)–H forms tight H– bonds with backbone amide carbonyls (MSOX: to Ileu50 backbone, 2.98 Å; GO: to Met49, 2.97 Å; DAAO: to Asp54, backbone 2.97 Å). The distribution of backbone amide C]O and N–H functionalities is thus very similar in GO and yeast DAAO as shown in Fig. 8. Also, in both cases there are ahelices with their positive ends pointing towards the pyrimidine ring of the flavin. In MSOX the presence and position of these groups and helices is maintained, however, there is an additional Lys-3-NHþ 3 group (K348) at a distance of 2.78 Å of the pyrimidine C(2)]O. This likely is a factor in the better stabilization of a negative charge on the flavin in MSOX, and provides a rationale for the low pKa z 8.3 for the ionization of oxidized flavin N(3)–H [8]. This argument has been discussed by Schuman Jorns and collaborators [38] who have verified the concept nicely by implementing appropriate mutations in MSOX. By comparison the oxidized flavin N(3)–H pKa in GO and yeast DAAO is slightly increased from that of free flavin (pKa z 10) [39]. Similarly affected is the pKa of the flavin radical that is <6 in MSOX [9] and >9 in GO (Fig. 2). As expected, with MSOX the Arg49 to Lys mutation induces an increase of z100 mV of the potential for the first electron transfer [38]. This should be reflected also in the Em of the three proteins, as is in general the case for such situations in flavoproteins and as discussed by Efimov and McIntire [40]. Indeed the E1 for GO and yeast DAAO are 120 and 140 mV [27,28] lower compared to that of MSOX. A peculiarity of GO is the presence of a water molecule at 2.95 Å from the flavin C(2)]O that forms H-bridges to Met49-C]O backbone (3.2 Å, Fig. 8). A water located in a similar position is found in yeast DAAO, but is not observed in MSOX (Fig. 8 top).
611
- Substrate binding: in all cases, i.e., with DAAO, GO and MSOX the substrate-COO group is linked at the active site via a double bridge to an arginine (Figs. 7 and 8). With DAAO two Tyr–OH groups (Tyr223 at 2.75 Å and Tyr238 at 3.1 Å) fix one carboxylate, while in GO only one (Tyr246, 3.9 Å) interacts, although less tightly with the same group. A role comparable to that of the ‘‘second Tyr’’ in DAAO is probably exerted in GO by Arg329 that is located at 2.9 Å in a corresponding position. As already mentioned above, this set-up is different in MSOX where substrate binding is mirrored with respect to the flavin N(5)–N(10) axis (Fig. 8). Also in this case a biforked interaction exists with Arg52 (3.1 Å) and a second one at 2.8 Å with Lys348 that would correspond to those exerted by the ‘‘second Tyr’’ and by the arginine residue mentioned above. Intriguingly, His345, Arg52, and Lys348 in MSOX form a network with 2–3 positive charges that probably constitute an important factor in determining the chemical properties of the flavin. The present comparisons do not evidentiate basic differences in the ‘‘set-up’’ of the three enzyme active sites. On the contrary, the differences appear to be gradual, this suggesting basic mechanistic similarities for the dehydrogenation of the two types of C–H substrates. It also appears that in all three cases multiple interactions concur in fixing the substrate in a strictly conserved position in which the C–H function that expels the hydride during dehydrogenation is placed on top of the flavin position N(5) (Fig. 8, see also discussion in [32]). On the other hand, the tuning of the flavin reactivity, as manifested by the interactions with the pyrimidine moiety, appears to reflect the requirement of different flavin redox potentials for dehydrogenation of the two types of substrates, the aC–H vs. the N–CH3 groups. Finally, on comparing the three active site centers (Figs. 7 and 8) one previously unnoticed feature
Fig. 8. Orientation of ligands, charges and functional groups at the active centers of GO, yeast DAAO and MSOX (see Fig. 7) [14,15,32,37]. The top row highlights the similar modes of ligand binding and fixation and the positioning of methionines in the active site. The bottom row evidences the oxygen (red) or nitrogen (blue) atoms, belonging to amide functions that encompass the flavin pyrimidine ring (van der Waals representation); in the case of MSOX, also the nitrogen of K348 side chain is highlighted.
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emerges: in the ‘‘upper part’’ of the active sites there are methionine side chains (Met49 in GO, Met213 in yeast DAAO, Met245 in MSOX) that stem from different segments of the polypeptide main chains, but are located within a distance of z6 Å. While this is unlikely to be coincidental, a functional role was made evident only for yeast DAAO, since Met213 plays an important role in the selection of the substrate based on its side chain [41]. 5. Conclusions
[14]
[15]
[16]
[17] [18]
In conclusion, the discussed differences in properties highlight how the substrate specificity, the flavin reactivity, and kinetic properties have been finely tuned starting from similar protein scaffolds to acquire different physiological roles. This information is also expected to be useful for the biotechnological use of GO since it defines the possible framework for such applications. For example, human DAAO can be fully inhibited by benzoate at a concentration at which GO is not (the Kd values for benzoate binding are at least two orders of magnitude lower for the human enzyme than for GO at all pH values) [42]. This, in turn, might make it possible to develop a glycine-sensitive biosensor based on GO (similar to the one recently developed for D-serine using yeast DAAO) [12], and whose response is not modified by the use of the DAAO inhibitors employed as drugs to treat complex mental diseases such as schizophrenia.
[19]
[20]
[21] [22] [23]
[24] [25]
Acknowledgments
[26]
This work was supported by grants from Fondo di Ateneo per la Ricerca to L. Pollegioni. We thank the support from Consorzio Interuniversitario per le Biotecnologie (CIB). This paper is dedicated to the memory of Prof. Stefano Ferrari.
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