- Email: [email protected]
[32] Methanol dehydrogenase from Hyphomicrobium X
202
METHYLOTROPHY
[32]
catalytic iron sites are provided by MOssbauer studies. Electron paramagnetic resonance (EPR) signals observed at g,v = 1.85 upon partial reduction and at g = 16 upon complete reduction are consistent with the presence of a p-oxo- or p-hydroxo-bridged binuclear iron cluster in the hydroxylase.4,1s The oxidized form of the hydroxylase exhibits an electronic absorption at 282 nm. No distinct optical features are observed above 300 nm. This is the first example of an oxygenase enzyme which appears to utilize an oxo-bridged binuclear iron cluster for oxygen activation chemistry. Cofactorsof Reductase. The oxidized reductase exhibits optical absorption maxima at 270, 340, 396, and 458 nm. Prominent shoulders are observed at 430 and 478 nm. The optical absorption smoothly decreases from 500 to 650 nm. In highly purified reductase preparations, the ratios of A27o/A458 and A458/A34o are 2.8 and 1.3, respectively. The reductase contains 1 mol of acid-dissociable FAD, 2 mol of iron, and 2 mol of inorganic sulfide per mole of protein. The observed ratio of iron to inorganic sulfide and the optical spectral properties are consistent with the presence of a [2Fe-2S] cluster in the reductase. Acknowledgments This work was supported by grants from the National Institutes of Health (GM 24689 and GM 40466).
[32] Methanol
Dehydrogenase
from
Hyphomicrobium X
By J. FRANK and J. A. DOINE CH3OH + dye ---*CH20 + dye • H2
Methanol dehydrogenase (EC 1.1.99.8) is a pyrroloquinoline quinone (PQQ)-containing oxidoreductase. The enzyme is involved in the oxidation of methanol by gram-negative methylotrophic bacteria.t It has a broad substrate specificity, and some facultative methylotrophs possibly use this enzyme for growth on higher alcohols. 2 Purification and (partial) characterization of the enzyme from Hyphomicrobium X has been reported previously.3 t C. Anthony, Adv. Microb. Physiol. 27, 113 (1986). 2 A. Groeneveld, M. Dijkstra, and J. A. Duine, FEMSMicrobiol. Lett. 25, 311 (1984). 3 j. A. Duine, J. Frank, and J. Westerling, Biochim. Biophys. Acta 524, 277 (1978).
METHODS IN ENZYMOLOGY,VOL. 188
Copyright@ 1990by AcademicPrim,Inc. All fightsofrtgroduetionin any formretziwed.
[32]
METHANOLDEHYDROGENASEFROM H y p h o m i c r o b i u m
203
Assay Methods
Principle.Activity measurements are based on the capabilityof certain cationic dyes to act as artificialelectron acceptors for enzyrnc reduced by methanol in the presence of activator (NH4CI) at high pH. Different dyes and different detection methods can be used. In Method I, activity is related to the amperometrically measured rate of oxygen consumption arisingfrom rcoxidation of reduced phenazine metho- or cthosulfate(PMS or PES). In Method 2, reduced P M S or PES is rcoxidized by dichlorophenolindophenol (DCPIP), and the rate of reduction of the secondary electron acceptor is measured spcctrophotometrically. Method 3 uses Wurster's blue, the perchlorate salt of the cationic free radical of N,N,N',N'-tetramethyl-p-phenylenediamine. PMS, PES and DCPIP are commercially available; Wurster's blue is not, but a convenient procedure for its synthesis has been described. 4 Both PMS and PES are inherently unstable, especially at high pH, leading to aldehyde production owing to dealkylation. These aldehydes are substrates with high affinity for the enzyme so that fresh dye solutions should be used since otherwise the difference between the blank and the measured activity becomes too small. In this respect PES is to be preferred over PMS as it is the more stable of the two, 5 but its Km value is 2 times higher. For the same reason, care should be exercised in preparing the solutions to avoid contamination with substrate (aldehydes are notorious contaminants in the laboratory atmosphere, and buffer salts are sometimes crystallized from alcoholic solutions). It should also be mentioned that enzyme preparations commonly contain varying amounts of an endogenous substrate of unknown nature? In principle, contaminating and endogenous substrate could be removed by preincubating the enzyme with electron acceptor, after which adequate measurements with the added substrate are possible. However, most methanol dehydrogenases are ,very labile under these conditions, that is, rapid inactivation occurs as soon as substrate is depleted. Cyanide is used in cases where enzyme preparations are still contaminated with components of the respiratory chain, blocking the catalysis of the reoxidation of reduced electron acceptor by oxygen. Cyanide also prevents the aforementioned inactivation of enzyme by electron acceptor and masks the effects of contaminating and endogenous substrate. This property is related to the competitive binding of cyanide to PQQ. Therefore, kinetic parameters should be obtained from measurements in the absence of cyanide. 4 L. Michaelisand S. Granick,J. Am. Chem. Soc. 65, 1747(1943). 5R. Gosh and J. R. Quayle,Anal Biochem. 99, 245 (1979).
204
METHYLOTROPHY
[32]
Reagents Sodium tetraborate buffer, 100 mM, pH 9.0, containing 2 m M methanol Potassium cyanide, 10 raM, pH 9.0 Ammonium chloride buffer, 500 raM, pH 9.0 Phenazine methosulfate or phenazine ethosulfate, 10 m M Dichlorophenolindophenol, 0.5 m M Wurster's blue, 5.25 m M Procedurefor Method 1. The assay is begun by placing 0.65 ml sodium tetraborate buffer, 0.1 ml ammonium chloride buffer, 0.1 ml potassium cyanide, and 0.1 ml phenazine methosulphate in the thermostatted cell (20*) of a biological oxygen monitor provided with a Clark electrode (Yellow Springs Instruments, Yellow Springs, OH). To this mixture 50/~1 of extract containing up to 50 milliunits of enzyme is added, and the rate of oxygen consumption is taken from the linear part of the curve. One unit is the amount of enzyme catalyzing the consumption of 1/~mol of oxygen per minute at 20 ° (assuming that H202 is formed and that no catalase is present). Procedurefor Method 2. To a cuvette (10-mm fight path) are added the following (in the indicated sequence): 0.55 ml sodium tetraborate buffer, 0.1 ml DCPIP, 0.1 ml potassium cyanide, 0.1 ml ammonium chloride buffer, 50/tl of extract, and 0.1 ml PMS or PES. After mixing the decrease in absorbance is followed at 600 nm, and the rate is calculated using an a600 value of 22,000 M-~ cm-~ for DCPIP. 6 One unit is the amount of enzyme catalyzing the reduction of 1/zmol of DCPIP per minute at 20 °. Procedurefor Method 3. To a cuvette (10-mm light path) are added the following (in the indicated sequence): 0.65 ml sodium tetraborate buffer, 0.1 ml potassium cyanide, 0.1 ml ammonium chloride buffer, and 0.1 ml Wurster's blue. After 30- 60 sec extract is added (50/zl), and the decrease in absorbance is followed at 640 nm. The rate is calculated using an E~0 value of 2143 M -~ cm -~ for Wurster's blue. 7 One unit is the amount of enzyme catalyzing the reduction of 2/~mol of Wurster's blue per minute at 20 ° (Wurster's blue is a one-electron acceptor). Purification P r o c e d u r e
Growth of Organism. Hyphomicrobium X, isolated by Attwood and Harder, 8 is grown at 30 ° on a mineral medium. 3 The basic medium 6 j. McD. Armstrong, Biochim. Biophys. Acta 86, 194 (1964). 7 L. Michaelis, M. P, Schubert, and S. Gramek, J. Am. Chem. Soc. 61, 1981 (1939). s M. M. Attwood and W. Harder, J. Microbiol. Serol. 38, 369 (1972).
[32]
METHANOLDEHYDROGENASEFROM Hyphomicrobium
205
contains, per liter, 2.28 g K2HPO 4- 3H20, 1.38 g N a H 2 P O 4. H 2 0 , 0.5 g (NH4)2SO4, and 0.2 g MgSO4" 7H20 and is brought to pH 7.0 with 2 M NaOH. After autoclaving, the following solutions are added to I liter of this medium: 1 ml of spore solution (sterilized by autoclaving), containing 7.8 mg C u S O 4 " 5 H 2 0 , 10 mg H3BO3, 10 mg MnSO4-4H20, 70 mg ZnSO4, and 10 mg MoO3 per liter, 1 ml of calcium solution (sterilized by autoclaving) containing 25 mg CaC12 • 2H20 per liter, 1 ml of iron solution (sterilized by filtration) containing 3.5 g FeC13"6H20 and 24.2 g tricine per liter, and 5 ml of methanol (sterilized by filtration). Growth in a fermentor (20 liter) is performed with an aeration of 7 liter/min and a stirrer speed of 200 rpm. The pH is maintained at 7.0 ___0.2 with 25% (v/v) ammonia solution. A second addition of methanol is made when the concentration of methanol falls below 0.1% (v/v) (as judged from gas chromatographic analysis a of the culture supernatant). The cells are harvested in the stationary phase by centrifugation at 48,000 g. Alternatively, growth is stopped by adjusting the pH of the culture to 4.0 (with concentrated HC1). The cells are allowed to settle by placing the fermentor in the cold room (4 °) for 3 hr. After decanting most of the clear supernatant, the cells are collected from the remaining suspension by centrifugation at 5000 g for 15 min. The cell paste is stored at - 2 0 °. All operations are carded out at room temperature, except dialysis and centrifugation which are performed at 4 °. Step 1: Preparation of Cell-Free Extract. Cell paste, 1000 g wet weight, is suspended in 1400 ml of 0.1 M Tris-HCl, 10 m M EDTA, pH 7.0. The suspension is mixed with 600 mg oflysozyme, allowed to stand for 10 min, and centrifuged at 27,300 g. The pellet is suspended in 1200 ml of 0.1 M Tris-HC1 containing 0.5% Triton X-100, pH 9.0, and, after standing at room temperature for 15 min, centrifuged as above. The pellet is reextracted in the same way and the supernatants are combined to give the cell-free extract. Step 2: Ammonium Sulfate Fractionation. Protein in the cell-free extract precipitating between 2.1 and 4.45 M (NH4)2SO4 (at 4 °) is collected by centrifugation at 13,000 g for 20 min; the pellet suspended in 200 ml of 20 m M potassium phosphate buffer, pH 7.0, and dialyzed overnight against 5 liters of the same buffer. Step 3: Silica Gel Treatment. The dialyzed enzyme preparation from Step 2 is centrifuged at 48,900 g for 15 min and passed through a column of silica gel (4 × 20 cm). The silica gel (Merck, type Si 60, 0.063-0.2 mm) is pretreated by heating at 550 ° for 1 hr. Just before use, the material is suspended in 20 m M potassium phosphate buffer, pH 7.0, and the mixture poured into the column. The cytochromes c are retained by the column while methanol dehydrogenase passes through and is washed from the
206
METHYLOTROPHY
[32]
column with 20 mM potassium phosphate buffer, pH 7.0. [Cytochrome CL and cytochrome CHcan be eluted with 20 m M potassium phosphate buffer, pH 7.0, containing 10% (w/v) polyethylene glycol 6000.] Step 4: Treatment with DEAE-Sepharose. After centrifugation of the pooled active fractions of Step 3 at 48,900 g for l0 min, the solution is applied to a DEAE-Sepharose (Fast How) column (4 X 15 cm), equilibrated with 20 mM potassium phosphate buffer, pH 7.0. The column is washed with the same buffer until all activity is eluted. Step 5: Hydroxyapatite Chromatography. The pooled fractions from Step 4 are applied to a hydroxyapatite column (4 × l0 cm), equilibrated with 20 mMpotassium phosphate buffer, pH 7.0. After washing with 4 bed volumes of 20 m M potassium phosphate buffer, pH 7.0 methanol dehydrogenase is eluted with 0.2 M potassium phosphate buffer, pH 7.0 (greenish-yellow fractions). Step 6: Phenyl-Sepharose Chromatography. Although the preparation obtained after Step 5 is usually pure, sometimes fluorescent contaminants are detected which can only be removed by including a chromatography step on Phenyl-Sepharose. For this, 200 mg of solid (NH+)2SO+ is added to 1 ml of the enzyme preparation of Step 5, and this solution is applied to a column of Phenyl-Sepharose (1 × 3 cm), equlibrated with 20 mM potassium phosphate buffer, pH 7.0, containing 1.5 M (NH4)2SO +. After washing the column with 3 bed volumes of the same buffer, containing 1.0 M (NH4)2SO4, methanol dehydrogenase is eluted with 10 m M sodium phosphate buffer, pH 7.0. The final methanol dehydrogenase preparation is sterilized by filtration (Acrodisc, Gelman, Ann Arbor, MI, 0.22 #m) and stored at + 4 °. The result of a typical purification is summarized in Table I. Properties
Purity. Polyacrylamide gel clcctrophoresis of the native enzyme shows a major and two minor bands which all display enzyme activity. Substrate Specificity. The enzyme oxidizes a large range of primary alcohols, as well as formaldehyde and acetaldehyde.9 Primary Electron Acceptors. Besides the phenazine derivatives and Wurster's blue, the free radical of 2,2'-azino-di-(3-ethylbenzthiazoline-6sulfonic acid) also accepts electrons from methanol dehydrogenase. Typically negatively charged electron acceptors (ferricyanide, DCPIP) are not effective. Activators. Activity depends on the presence of ammonium chloride. It 9 j. A. Duine and J. Frank, Biochem. J. 187, 213 (1980).
[32]
METHANOLDEHYDROGENASEFROM Hyphomicrobium
207
TABLE I PURIFICATIONOF METHANOLDEHYDROGENASEFROMHyphomicrobium X
Fraction Step 1. Cell-free extract Step 2. (NH4)2SO4 fraction (2.1-4.45 M) Step 3. Silica gel percolate Step 4. DEAE-Sepharose percolate Step 5. Hydroxyapatite eluate Step 6. Phenyl-Sepharose eluate
Specific activity (units/mg protein)
Yield (%)
7427 5073
0.3 0.8
100 68
5764 1411
5073 3750
0.9 2.7
68 50
985 926
3088 2941
3.1 3.2
42 40
Total volume (ml)
Total protein (mg)
Total activity" (units)
2353 327
24,941 6397
398 485 220 294
a Results obtained with Assay Method 3.
can be replaced by benzyl and ethyl esters of glycine, phenylpropylamine, and phenylbutyramine, substituted benzylamines, and 2-bromoethylamine. 1° Methylamine is a poor activator. Inhibitors. Cyanide (kl 1 raM) and hydroxylamine (ki 12/zM) are competitive inhibitors with respect to methanol. 9 Cyclopropanol and cyclopropanone hydrate irreversibly and stoichiometrically (with respect to PQQ) inhibit the enzyme) ~ Irreversible inhibition has also been observed with methylhydrazine. ~o Absorption Coefficients. Using the method of van Iersel et al., 12 ,,,~-,~28o~°'~ of 2.04 has been determined. Molar absorption coet~cients can be derived from this value for the three different redox forms of the enzyme: Eago=20,400M -~ cm -1 for the oxidized e n z y m e - H C N complex (MDHox-HCN), E343=38,000M -1 cm -1 for the reduced enzyme ( M D H ~ ) , and E~s -- 29,000 M -1 cm -1 is for the semiquinone form (MDH~m). Redox Forms. The purification scheme as presented here yields the MDH,m form as judged from the absorption spectrum (Fig. 1) and the free radical present in the preparation (Fig. 2). This form of the enzyme does not react with the alcohol substrate. It can be reduced photochemically with EDTA and deazalumiflavine 13 or with the methyl viologen cation radical to M D H ~ 14 (Fig. 1). Oxidation with artificial electron acceptors 1oj. A. Duine and J. Frank, unpublished results (1980). 11 M. Dijkstra, J. Frank, J. A. Jongejan, and J. A. Dulne, Eur. J. Biochern. 140, 369 (1984). 12j. van Iersel, J. Frank, and J. A. Dulne, Anal Biochem. 151, 196 (1985). 13j. Frank, M. Dijkstra, J. A. Duine, and C. Balny, Eur. J. Biochem. 174, 331 (1988). 14R. de Beer, J. A. Duine, J. Frank, and J. Westerling, Eur. J. Biochem. 130, 105 (1983).
208
[32]
METHYLOTROPHY 1.0
c
0.8
~
0.6
r~ _Q
0.4
-.../ / --_ . ...... .
\
"-,
/
\ \
"'-.\
0.2 0.0
300
l
[
350
4-00
450
Wavelength (nm) FIG. 1. Absorption spectra of the different redox forms of methanol dehydrogenase. MDH~ ( ), MDH~m (---), and MDHox"HCN ( - - - ) , all at a concentration of 22 gait in 20 mM potassium phosphate buffer, pH 7.0.
resultsin the formation of M D H o ~ w h i c h is only stable in the presence of a competitive inhibitor such as cyanide or hydroxylaminc, 9 owing to adduct formation of these compounds with thc cofactor. U p o n oxidation in the presence of substratc (but without activator)a transient oxidized enzymcsubstratc complex (MDHox. S) can bc observed? 3
. O)
= (D
/
b
tw 3 mT FIG. 2. X-band electron spin resonance spectra of methanol dehydrogenase. The spectrum is shown of MDH~min air-saturated buffer after cooling to liquid nitrogen temperature (a). The signals in spectrum a, indicated with arrows, do not appear when the solution is flushed with nitrogen prior to cooling (b).
[32]
METHANOLDEHYDROGENASEFROM Hyphomicrobium
209
Apoenzyme. In all attempts to reconstitute PQQ-depleted enzyme to holoenzyme, activity has not been observed. 1°,~5 Molecular Weight, Subunit Structure, and Cofactor Content. A molecular weight of 120,000 is found upon gel-permeation chromatography on Sephadex G-200. 9 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows two bands with molecular weights of 60,000 and 8,000,1° indicating that the enzyme consists of four subunits. Two molecules of PQQ per enzyme molecule la can be extracted ~6 from the enzyme. Kinetic Parameters. With Wurster's blue as the electron acceptor (50 m M NH4C1 as activator, Method 3) an apparent Km of 0.3 m M is found for methanol. Methanol oxidation in the ES complex can be described by a single exponential and is fully rate limiting (k 0.06 sec-~) in the absence of activator and only partly rate limiting (k 23 sec-~) in its presence. ~a Oxidation of M D H ~ and MDH~.~ by Wurster's blue is rapid at pH 9.0 (k 2.22 × 105 and 0.75 × 105 M -~ sec -~, respectively) and slow at pH 7.0 (k 3291 and 2280 M -t sec-l), la Natural Electron Acceptor and Activator. In contrast to artificial electron acceptors, the natural one, cytochrome CL, very actively oxidizes M D H ~ and MDH~m at pH 7.0 (k 1.9 × 105 and 2.1 × 105 M -1 sec-1, respectively17), but not at pH 9.0. Autoreduction Is of this cytochrome does not play a role in this process 19 (autoreduction is negligible when the cytochrome is purified on Phenyl-Sepharosel7). An oxygen-sensitive compound has been detected which is most probably the natural activator as it activates the substrate oxidation step at pH 7.020 (NH4CI has a very low activity at this pH). Stability and pH Optimum. Storage of the enzyme preparations in frozen form induces changes in the absorption spectrum, ~4and significant loss of activity occurs. This might be related to the interaction of 02 with PQQH" at low temperatures as can be observed in the electron spin resonance (ESR) spectrum ~4 (Fig. 2). For these reasons, sterile storage at 4 ° is recommended (loss of activity is not more than 30% per year in 0.2 M potassium phosphate buffer, pH 7.0).
~ V. L. Davidson, J. W. Neher, and G. Cecchini, J. Biol. Chem. 17, 9642 (1985). 16 R. A. van der Meer, B. W. Groen, M. A. G. van Kleef, J. Frank, J. A. Jongejan, and J. A. Duine, this volume [41 ]. 17 M. Dijkstra, J. Frank, and J. A. Duine, Biochem. J. 257, 87 (1989). is D. T. O'Keefe and C. Anthony, Biochem. J. 190, 481 (1980). ~9M. Dijkstra, J. Frank, J. E. van Wielink, and J. A. Duine, Biochem. J. 251,467 (1988). 20 M. Dijkstra, J. Frank, and J. A. Duine, FEBSLett. 227, 198 (1988).
METHYLOTROPHY
[32]
catalytic iron sites are provided by MOssbauer studies. Electron paramagnetic resonance (EPR) signals observed at g,v = 1.85 upon partial reduction and at g = 16 upon complete reduction are consistent with the presence of a p-oxo- or p-hydroxo-bridged binuclear iron cluster in the hydroxylase.4,1s The oxidized form of the hydroxylase exhibits an electronic absorption at 282 nm. No distinct optical features are observed above 300 nm. This is the first example of an oxygenase enzyme which appears to utilize an oxo-bridged binuclear iron cluster for oxygen activation chemistry. Cofactorsof Reductase. The oxidized reductase exhibits optical absorption maxima at 270, 340, 396, and 458 nm. Prominent shoulders are observed at 430 and 478 nm. The optical absorption smoothly decreases from 500 to 650 nm. In highly purified reductase preparations, the ratios of A27o/A458 and A458/A34o are 2.8 and 1.3, respectively. The reductase contains 1 mol of acid-dissociable FAD, 2 mol of iron, and 2 mol of inorganic sulfide per mole of protein. The observed ratio of iron to inorganic sulfide and the optical spectral properties are consistent with the presence of a [2Fe-2S] cluster in the reductase. Acknowledgments This work was supported by grants from the National Institutes of Health (GM 24689 and GM 40466).
[32] Methanol
Dehydrogenase
from
Hyphomicrobium X
By J. FRANK and J. A. DOINE CH3OH + dye ---*CH20 + dye • H2
Methanol dehydrogenase (EC 1.1.99.8) is a pyrroloquinoline quinone (PQQ)-containing oxidoreductase. The enzyme is involved in the oxidation of methanol by gram-negative methylotrophic bacteria.t It has a broad substrate specificity, and some facultative methylotrophs possibly use this enzyme for growth on higher alcohols. 2 Purification and (partial) characterization of the enzyme from Hyphomicrobium X has been reported previously.3 t C. Anthony, Adv. Microb. Physiol. 27, 113 (1986). 2 A. Groeneveld, M. Dijkstra, and J. A. Duine, FEMSMicrobiol. Lett. 25, 311 (1984). 3 j. A. Duine, J. Frank, and J. Westerling, Biochim. Biophys. Acta 524, 277 (1978).
METHODS IN ENZYMOLOGY,VOL. 188
Copyright@ 1990by AcademicPrim,Inc. All fightsofrtgroduetionin any formretziwed.
[32]
METHANOLDEHYDROGENASEFROM H y p h o m i c r o b i u m
203
Assay Methods
Principle.Activity measurements are based on the capabilityof certain cationic dyes to act as artificialelectron acceptors for enzyrnc reduced by methanol in the presence of activator (NH4CI) at high pH. Different dyes and different detection methods can be used. In Method I, activity is related to the amperometrically measured rate of oxygen consumption arisingfrom rcoxidation of reduced phenazine metho- or cthosulfate(PMS or PES). In Method 2, reduced P M S or PES is rcoxidized by dichlorophenolindophenol (DCPIP), and the rate of reduction of the secondary electron acceptor is measured spcctrophotometrically. Method 3 uses Wurster's blue, the perchlorate salt of the cationic free radical of N,N,N',N'-tetramethyl-p-phenylenediamine. PMS, PES and DCPIP are commercially available; Wurster's blue is not, but a convenient procedure for its synthesis has been described. 4 Both PMS and PES are inherently unstable, especially at high pH, leading to aldehyde production owing to dealkylation. These aldehydes are substrates with high affinity for the enzyme so that fresh dye solutions should be used since otherwise the difference between the blank and the measured activity becomes too small. In this respect PES is to be preferred over PMS as it is the more stable of the two, 5 but its Km value is 2 times higher. For the same reason, care should be exercised in preparing the solutions to avoid contamination with substrate (aldehydes are notorious contaminants in the laboratory atmosphere, and buffer salts are sometimes crystallized from alcoholic solutions). It should also be mentioned that enzyme preparations commonly contain varying amounts of an endogenous substrate of unknown nature? In principle, contaminating and endogenous substrate could be removed by preincubating the enzyme with electron acceptor, after which adequate measurements with the added substrate are possible. However, most methanol dehydrogenases are ,very labile under these conditions, that is, rapid inactivation occurs as soon as substrate is depleted. Cyanide is used in cases where enzyme preparations are still contaminated with components of the respiratory chain, blocking the catalysis of the reoxidation of reduced electron acceptor by oxygen. Cyanide also prevents the aforementioned inactivation of enzyme by electron acceptor and masks the effects of contaminating and endogenous substrate. This property is related to the competitive binding of cyanide to PQQ. Therefore, kinetic parameters should be obtained from measurements in the absence of cyanide. 4 L. Michaelisand S. Granick,J. Am. Chem. Soc. 65, 1747(1943). 5R. Gosh and J. R. Quayle,Anal Biochem. 99, 245 (1979).
204
METHYLOTROPHY
[32]
Reagents Sodium tetraborate buffer, 100 mM, pH 9.0, containing 2 m M methanol Potassium cyanide, 10 raM, pH 9.0 Ammonium chloride buffer, 500 raM, pH 9.0 Phenazine methosulfate or phenazine ethosulfate, 10 m M Dichlorophenolindophenol, 0.5 m M Wurster's blue, 5.25 m M Procedurefor Method 1. The assay is begun by placing 0.65 ml sodium tetraborate buffer, 0.1 ml ammonium chloride buffer, 0.1 ml potassium cyanide, and 0.1 ml phenazine methosulphate in the thermostatted cell (20*) of a biological oxygen monitor provided with a Clark electrode (Yellow Springs Instruments, Yellow Springs, OH). To this mixture 50/~1 of extract containing up to 50 milliunits of enzyme is added, and the rate of oxygen consumption is taken from the linear part of the curve. One unit is the amount of enzyme catalyzing the consumption of 1/~mol of oxygen per minute at 20 ° (assuming that H202 is formed and that no catalase is present). Procedurefor Method 2. To a cuvette (10-mm fight path) are added the following (in the indicated sequence): 0.55 ml sodium tetraborate buffer, 0.1 ml DCPIP, 0.1 ml potassium cyanide, 0.1 ml ammonium chloride buffer, 50/tl of extract, and 0.1 ml PMS or PES. After mixing the decrease in absorbance is followed at 600 nm, and the rate is calculated using an a600 value of 22,000 M-~ cm-~ for DCPIP. 6 One unit is the amount of enzyme catalyzing the reduction of 1/zmol of DCPIP per minute at 20 °. Procedurefor Method 3. To a cuvette (10-mm light path) are added the following (in the indicated sequence): 0.65 ml sodium tetraborate buffer, 0.1 ml potassium cyanide, 0.1 ml ammonium chloride buffer, and 0.1 ml Wurster's blue. After 30- 60 sec extract is added (50/zl), and the decrease in absorbance is followed at 640 nm. The rate is calculated using an E~0 value of 2143 M -~ cm -~ for Wurster's blue. 7 One unit is the amount of enzyme catalyzing the reduction of 2/~mol of Wurster's blue per minute at 20 ° (Wurster's blue is a one-electron acceptor). Purification P r o c e d u r e
Growth of Organism. Hyphomicrobium X, isolated by Attwood and Harder, 8 is grown at 30 ° on a mineral medium. 3 The basic medium 6 j. McD. Armstrong, Biochim. Biophys. Acta 86, 194 (1964). 7 L. Michaelis, M. P, Schubert, and S. Gramek, J. Am. Chem. Soc. 61, 1981 (1939). s M. M. Attwood and W. Harder, J. Microbiol. Serol. 38, 369 (1972).
[32]
METHANOLDEHYDROGENASEFROM Hyphomicrobium
205
contains, per liter, 2.28 g K2HPO 4- 3H20, 1.38 g N a H 2 P O 4. H 2 0 , 0.5 g (NH4)2SO4, and 0.2 g MgSO4" 7H20 and is brought to pH 7.0 with 2 M NaOH. After autoclaving, the following solutions are added to I liter of this medium: 1 ml of spore solution (sterilized by autoclaving), containing 7.8 mg C u S O 4 " 5 H 2 0 , 10 mg H3BO3, 10 mg MnSO4-4H20, 70 mg ZnSO4, and 10 mg MoO3 per liter, 1 ml of calcium solution (sterilized by autoclaving) containing 25 mg CaC12 • 2H20 per liter, 1 ml of iron solution (sterilized by filtration) containing 3.5 g FeC13"6H20 and 24.2 g tricine per liter, and 5 ml of methanol (sterilized by filtration). Growth in a fermentor (20 liter) is performed with an aeration of 7 liter/min and a stirrer speed of 200 rpm. The pH is maintained at 7.0 ___0.2 with 25% (v/v) ammonia solution. A second addition of methanol is made when the concentration of methanol falls below 0.1% (v/v) (as judged from gas chromatographic analysis a of the culture supernatant). The cells are harvested in the stationary phase by centrifugation at 48,000 g. Alternatively, growth is stopped by adjusting the pH of the culture to 4.0 (with concentrated HC1). The cells are allowed to settle by placing the fermentor in the cold room (4 °) for 3 hr. After decanting most of the clear supernatant, the cells are collected from the remaining suspension by centrifugation at 5000 g for 15 min. The cell paste is stored at - 2 0 °. All operations are carded out at room temperature, except dialysis and centrifugation which are performed at 4 °. Step 1: Preparation of Cell-Free Extract. Cell paste, 1000 g wet weight, is suspended in 1400 ml of 0.1 M Tris-HCl, 10 m M EDTA, pH 7.0. The suspension is mixed with 600 mg oflysozyme, allowed to stand for 10 min, and centrifuged at 27,300 g. The pellet is suspended in 1200 ml of 0.1 M Tris-HC1 containing 0.5% Triton X-100, pH 9.0, and, after standing at room temperature for 15 min, centrifuged as above. The pellet is reextracted in the same way and the supernatants are combined to give the cell-free extract. Step 2: Ammonium Sulfate Fractionation. Protein in the cell-free extract precipitating between 2.1 and 4.45 M (NH4)2SO4 (at 4 °) is collected by centrifugation at 13,000 g for 20 min; the pellet suspended in 200 ml of 20 m M potassium phosphate buffer, pH 7.0, and dialyzed overnight against 5 liters of the same buffer. Step 3: Silica Gel Treatment. The dialyzed enzyme preparation from Step 2 is centrifuged at 48,900 g for 15 min and passed through a column of silica gel (4 × 20 cm). The silica gel (Merck, type Si 60, 0.063-0.2 mm) is pretreated by heating at 550 ° for 1 hr. Just before use, the material is suspended in 20 m M potassium phosphate buffer, pH 7.0, and the mixture poured into the column. The cytochromes c are retained by the column while methanol dehydrogenase passes through and is washed from the
206
METHYLOTROPHY
[32]
column with 20 mM potassium phosphate buffer, pH 7.0. [Cytochrome CL and cytochrome CHcan be eluted with 20 m M potassium phosphate buffer, pH 7.0, containing 10% (w/v) polyethylene glycol 6000.] Step 4: Treatment with DEAE-Sepharose. After centrifugation of the pooled active fractions of Step 3 at 48,900 g for l0 min, the solution is applied to a DEAE-Sepharose (Fast How) column (4 X 15 cm), equilibrated with 20 mM potassium phosphate buffer, pH 7.0. The column is washed with the same buffer until all activity is eluted. Step 5: Hydroxyapatite Chromatography. The pooled fractions from Step 4 are applied to a hydroxyapatite column (4 × l0 cm), equilibrated with 20 mMpotassium phosphate buffer, pH 7.0. After washing with 4 bed volumes of 20 m M potassium phosphate buffer, pH 7.0 methanol dehydrogenase is eluted with 0.2 M potassium phosphate buffer, pH 7.0 (greenish-yellow fractions). Step 6: Phenyl-Sepharose Chromatography. Although the preparation obtained after Step 5 is usually pure, sometimes fluorescent contaminants are detected which can only be removed by including a chromatography step on Phenyl-Sepharose. For this, 200 mg of solid (NH+)2SO+ is added to 1 ml of the enzyme preparation of Step 5, and this solution is applied to a column of Phenyl-Sepharose (1 × 3 cm), equlibrated with 20 mM potassium phosphate buffer, pH 7.0, containing 1.5 M (NH4)2SO +. After washing the column with 3 bed volumes of the same buffer, containing 1.0 M (NH4)2SO4, methanol dehydrogenase is eluted with 10 m M sodium phosphate buffer, pH 7.0. The final methanol dehydrogenase preparation is sterilized by filtration (Acrodisc, Gelman, Ann Arbor, MI, 0.22 #m) and stored at + 4 °. The result of a typical purification is summarized in Table I. Properties
Purity. Polyacrylamide gel clcctrophoresis of the native enzyme shows a major and two minor bands which all display enzyme activity. Substrate Specificity. The enzyme oxidizes a large range of primary alcohols, as well as formaldehyde and acetaldehyde.9 Primary Electron Acceptors. Besides the phenazine derivatives and Wurster's blue, the free radical of 2,2'-azino-di-(3-ethylbenzthiazoline-6sulfonic acid) also accepts electrons from methanol dehydrogenase. Typically negatively charged electron acceptors (ferricyanide, DCPIP) are not effective. Activators. Activity depends on the presence of ammonium chloride. It 9 j. A. Duine and J. Frank, Biochem. J. 187, 213 (1980).
[32]
METHANOLDEHYDROGENASEFROM Hyphomicrobium
207
TABLE I PURIFICATIONOF METHANOLDEHYDROGENASEFROMHyphomicrobium X
Fraction Step 1. Cell-free extract Step 2. (NH4)2SO4 fraction (2.1-4.45 M) Step 3. Silica gel percolate Step 4. DEAE-Sepharose percolate Step 5. Hydroxyapatite eluate Step 6. Phenyl-Sepharose eluate
Specific activity (units/mg protein)
Yield (%)
7427 5073
0.3 0.8
100 68
5764 1411
5073 3750
0.9 2.7
68 50
985 926
3088 2941
3.1 3.2
42 40
Total volume (ml)
Total protein (mg)
Total activity" (units)
2353 327
24,941 6397
398 485 220 294
a Results obtained with Assay Method 3.
can be replaced by benzyl and ethyl esters of glycine, phenylpropylamine, and phenylbutyramine, substituted benzylamines, and 2-bromoethylamine. 1° Methylamine is a poor activator. Inhibitors. Cyanide (kl 1 raM) and hydroxylamine (ki 12/zM) are competitive inhibitors with respect to methanol. 9 Cyclopropanol and cyclopropanone hydrate irreversibly and stoichiometrically (with respect to PQQ) inhibit the enzyme) ~ Irreversible inhibition has also been observed with methylhydrazine. ~o Absorption Coefficients. Using the method of van Iersel et al., 12 ,,,~-,~28o~°'~ of 2.04 has been determined. Molar absorption coet~cients can be derived from this value for the three different redox forms of the enzyme: Eago=20,400M -~ cm -1 for the oxidized e n z y m e - H C N complex (MDHox-HCN), E343=38,000M -1 cm -1 for the reduced enzyme ( M D H ~ ) , and E~s -- 29,000 M -1 cm -1 is for the semiquinone form (MDH~m). Redox Forms. The purification scheme as presented here yields the MDH,m form as judged from the absorption spectrum (Fig. 1) and the free radical present in the preparation (Fig. 2). This form of the enzyme does not react with the alcohol substrate. It can be reduced photochemically with EDTA and deazalumiflavine 13 or with the methyl viologen cation radical to M D H ~ 14 (Fig. 1). Oxidation with artificial electron acceptors 1oj. A. Duine and J. Frank, unpublished results (1980). 11 M. Dijkstra, J. Frank, J. A. Jongejan, and J. A. Dulne, Eur. J. Biochern. 140, 369 (1984). 12j. van Iersel, J. Frank, and J. A. Dulne, Anal Biochem. 151, 196 (1985). 13j. Frank, M. Dijkstra, J. A. Duine, and C. Balny, Eur. J. Biochem. 174, 331 (1988). 14R. de Beer, J. A. Duine, J. Frank, and J. Westerling, Eur. J. Biochem. 130, 105 (1983).
208
[32]
METHYLOTROPHY 1.0
c
0.8
~
0.6
r~ _Q
0.4
-.../ / --_ . ...... .
\
"-,
/
\ \
"'-.\
0.2 0.0
300
l
[
350
4-00
450
Wavelength (nm) FIG. 1. Absorption spectra of the different redox forms of methanol dehydrogenase. MDH~ ( ), MDH~m (---), and MDHox"HCN ( - - - ) , all at a concentration of 22 gait in 20 mM potassium phosphate buffer, pH 7.0.
resultsin the formation of M D H o ~ w h i c h is only stable in the presence of a competitive inhibitor such as cyanide or hydroxylaminc, 9 owing to adduct formation of these compounds with thc cofactor. U p o n oxidation in the presence of substratc (but without activator)a transient oxidized enzymcsubstratc complex (MDHox. S) can bc observed? 3
. O)
= (D
/
b
tw 3 mT FIG. 2. X-band electron spin resonance spectra of methanol dehydrogenase. The spectrum is shown of MDH~min air-saturated buffer after cooling to liquid nitrogen temperature (a). The signals in spectrum a, indicated with arrows, do not appear when the solution is flushed with nitrogen prior to cooling (b).
[32]
METHANOLDEHYDROGENASEFROM Hyphomicrobium
209
Apoenzyme. In all attempts to reconstitute PQQ-depleted enzyme to holoenzyme, activity has not been observed. 1°,~5 Molecular Weight, Subunit Structure, and Cofactor Content. A molecular weight of 120,000 is found upon gel-permeation chromatography on Sephadex G-200. 9 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows two bands with molecular weights of 60,000 and 8,000,1° indicating that the enzyme consists of four subunits. Two molecules of PQQ per enzyme molecule la can be extracted ~6 from the enzyme. Kinetic Parameters. With Wurster's blue as the electron acceptor (50 m M NH4C1 as activator, Method 3) an apparent Km of 0.3 m M is found for methanol. Methanol oxidation in the ES complex can be described by a single exponential and is fully rate limiting (k 0.06 sec-~) in the absence of activator and only partly rate limiting (k 23 sec-~) in its presence. ~a Oxidation of M D H ~ and MDH~.~ by Wurster's blue is rapid at pH 9.0 (k 2.22 × 105 and 0.75 × 105 M -~ sec -~, respectively) and slow at pH 7.0 (k 3291 and 2280 M -t sec-l), la Natural Electron Acceptor and Activator. In contrast to artificial electron acceptors, the natural one, cytochrome CL, very actively oxidizes M D H ~ and MDH~m at pH 7.0 (k 1.9 × 105 and 2.1 × 105 M -1 sec-1, respectively17), but not at pH 9.0. Autoreduction Is of this cytochrome does not play a role in this process 19 (autoreduction is negligible when the cytochrome is purified on Phenyl-Sepharosel7). An oxygen-sensitive compound has been detected which is most probably the natural activator as it activates the substrate oxidation step at pH 7.020 (NH4CI has a very low activity at this pH). Stability and pH Optimum. Storage of the enzyme preparations in frozen form induces changes in the absorption spectrum, ~4and significant loss of activity occurs. This might be related to the interaction of 02 with PQQH" at low temperatures as can be observed in the electron spin resonance (ESR) spectrum ~4 (Fig. 2). For these reasons, sterile storage at 4 ° is recommended (loss of activity is not more than 30% per year in 0.2 M potassium phosphate buffer, pH 7.0).
~ V. L. Davidson, J. W. Neher, and G. Cecchini, J. Biol. Chem. 17, 9642 (1985). 16 R. A. van der Meer, B. W. Groen, M. A. G. van Kleef, J. Frank, J. A. Jongejan, and J. A. Duine, this volume [41 ]. 17 M. Dijkstra, J. Frank, and J. A. Duine, Biochem. J. 257, 87 (1989). is D. T. O'Keefe and C. Anthony, Biochem. J. 190, 481 (1980). ~9M. Dijkstra, J. Frank, J. E. van Wielink, and J. A. Duine, Biochem. J. 251,467 (1988). 20 M. Dijkstra, J. Frank, and J. A. Duine, FEBSLett. 227, 198 (1988).