Identification of the covalent flavin adenine dinucleotide-binding region in pyranose 2-oxidase from Trametes multicolor

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 314 (2003) 235–242 www.elsevier.com/locate/yabio

Identification of the covalent flavin adenine dinucleotide-binding region in pyranose 2-oxidase from Trametes multicolor Petr Halada,a,* Christian Leitner,b Petr Sedmera,a Dietmar Haltrich,b and Jind
rich Volca b

a Institute of Microbiology, Academy of Sciences of the Czech Republic, Vıde
nsk a 1083, CZ-142 20 Prague 4, Czech Republic Division of Biochemical Engineering, Institute of Food Technology, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria

Received 12 August 2002

Abstract We present the first report on characterization of the covalent flavinylation site in flavoprotein pyranose 2-oxidase. Pyranose 2oxidase from the basidiomycete fungus Trametes multicolor, catalyzing C-2/C-3 oxidation of several monosaccharides, shows typical absorption maxima of flavoproteins at 456, 345, and 275 nm. No release of flavin was observed after protein denaturation, indicating covalent attachment of the cofactor. The flavopeptide fragment resulting from tryptic/chymotryptic digestion of the purified enzyme was isolated by anion-exchange and reversed-phase high-performance liquid chromatography. The flavin type, attachment site, and mode of its linkage were determined by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy of the intact flavopeptide, without its prior enzymatic degradation to the central aminoacyl moiety. Mass spectrometry identified the attached flavin as flavin adenine dinucleotide (FAD). Post-source decay analysis revealed that the flavin is covalently bound to histidine residue in the peptide STHW, consistent with the results of N-terminal amino acid sequencing by Edman degradation. The type of the aminoacyl flavin covalent link was determined by NMR spectroscopy, resulting in the structure 8a-(N 3 -histidyl)-FAD. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Flavoprotein; Flavinylation site; MALDI mass spectrometry; Pyranose 2-oxidase; Covalent flavopeptide; NMR spectroscopy

Pyranose 2-oxidase (P2O;1 pyranose:oxygen 2-oxidoreductase, EC 1.1.3.10), a fungal periplasmic homotetrameric flavoprotein (
300 kDa), has received increased attention due to its potential analytical and biotechnological applications [1]. This enzyme catalyzes C-2/C-3 oxidation of numerous sugars to their corresponding dicarbonyl derivatives (aldos-2-uloses or gly-

*

Corresponding author. Fax: +420-2-41062749. E-mail address: [email protected] (P. Halada). 1 Abbreviations used: CID, collisionally induced dissociation; 1DTOCSY, one-dimensional total correlation spectroscopy; ESI, electrospray ionization; HMBC, heteronuclear multiple bond correlation; HMQC, heteronuclear multiple quantum correlation; LR COSY, long-range correlated spectroscopy; MALDI, matrix-assisted laser desorption/ionization; P2O, pyranose 2-oxidase; PSD, post-source decay; ROESY, rotating frame Overhauser enhancement spectroscopy; FNM, flavin mononucleotide; TFA, trifluoroacetic acid; 2D, two-dimensional; PTH, phenylthiohydantoin.

cosid-3-uloses), coupled to the reduction of FAD, an obligatory cofactor. The comprehensive knowledge of P2O biochemistry has been reviewed recently [2]. Despite P2OÕs suggested role in ligninolytic system(s) of some wood-degrading fungi and its applied aspects, the molecular structure of this enzyme is poorly understood. The primary structure is known for P2O from Trametes (Coriolus) versicolor [3] and T. hirsuta [4] as deduced from the nucleotide sequence of p2o cDNA. T. versicolor P2O is obviously synthesized as a preproenzyme containing 623 amino acids, which is localized in the periplasmic space after 38 amino acid processing. The mature enzyme (calculated subunit Mr ¼ 65;547Þ is apparently not glycosylated and contains one covalently bound FAD molecule per each subunit [5,6]. T. hirsuta preproenzyme P2O consists of 622 amino acids, the first 27 of them probably comprising a secretory signal peptide. The sequences of the two P2Os exhibit 84% identity. Additionally, based on

0003-2697/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(02)00661-9

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peptide mass mapping and PSD analysis on MALDIMS, we have recently found at least 54% identity of T. multicolor and T. versicolor P2O [7]. The mentioned P2Os include a highly conserved sequence motif Gly-XGly-X-X-Gly, which was shown to be involved in initial noncovalent interaction with the ADP moiety of FAD [8]. However, no attempts were made to determine the site and mode of the FAD covalent attachment to the P2O polypeptide. Although covalent flavoenzymes are less widespread (approx. 30 members to date) than their counterparts containing dissociable FAD/FMN cofactor, considerable knowledge has already been accumulated with regard to their structural features [9,10]. The data show significant diversity in the mode of flavin redox center incorporation in protein structure. In general, three distinct types of flavin attachment to the polypeptide chain have been recognized: at the FMN phosphate group via a threonyl (O) residue [11,12], the C-6 of FMN isoalloxazine ring via a cysteinyl (S) residue, and, most frequently, the 8a-methyl group of FAD or FMN isoalloxazine ring through the variable, albeit protein-specific, residues, cysteinyl (S), tyrosyl (O), and histidyl (N 1 or N 3 ) [9]. The subjects of current discussion are whether there are specific sequence motifs for the individual modes of flavin linkage and the nature of the general mechanisms of the multistep self-catalytic process proposed for flavin covalent attachment [13]. The aim of our research was to determine the structure of the flavopeptide purified from a proteolytic digest of P2O produced by the fungus T. multicolor, to characterize and classify this enzyme in terms of its flavin species. A methodological strategy based on MS and NMR techniques was utilized to fully characterize the isolated flavinylated peptide without submitting it to subsequent enzymatic degradation to the level of the aminoacyl riboflavin central moiety employed in conventional approaches [10]. MS analysis identified flavopeptide sequence, flavin type, and flavin linkage site. NMR confirmed the presence of Ser, Thr, His, Trp, and 8a-modified FAD. Importantly, it provided an experimental corroboration for the attachment of the flavin 8a-CH2 to N 3 of histidine residue in the sequence STHW. The data obtained are discussed in the context of flavinylated peptide sequences reported for other covalent flavoproteins.

Materials and methods Flavoprotein purification and proteolysis Pyranose 2-oxidase from T. multicolor was obtained from a 20-L fermentation [14] and purified to apparent homogeneity as described recently [15]. The en-

zyme (467 mg) in 50 mM potassium phosphate, pH 6.5 (30 ml), was heated to 95 °C for 10 min, cooled to room temperature, and centrifuged for 20 min at 10,000g. After washing twice with 0.2 M NH4 HCO3 , the precipitate was resuspended in 30 ml of the same solution. The suspension was supplemented with trypsin/chymotrypsin (Promega, Madison, WI; 30 mg each) and incubated at 38 °C under stirring. The pH was maintained at 8.0 by titration with 1 M acetic acid. After the precipitate was dissolved (30 min), the proteolysis was monitored by reversed-phase HPLC on a Supelco LC-318 column. The reaction was stopped after 8 h. Purification of FAD peptide The peptide mixture was brought to pH 6.0 with 1 M acetic acid and the formed precipitate removed by filtration through a 0.45-lm filter. The clear filtrate was degassed and loaded on a Source 30Q column (20 ml) preequilibrated with 20 mM bisTrisHCl, pH 6.0 (buffer A). Subsequently, peptides were eluted using a linear gradient of 0–40% 0.5 M NaCl in buffer A (600 ml) and absorption of the eluate was measured at 220 and 450 nm. Fractions with increased absorbance at 450 nm were pooled and run in two aliquots on a Supelcosil SPLC LC-18-DB column preequilibrated with 0.1% TFA. A linear gradient (0–100%) of 60% acetonitrile in 0.1% TFA (800 ml) was applied at 50 °C. The flavopeptide-containing fraction was freeze-dried (5 mg) and stored at )20 °C. FAD content was calculated based on the molar extinction coefficient of free FAD 460 ¼ 11:3 mM1 cm1 [16]. MALDI mass spectrometry A saturated solution of a-cyano-4-hydroxycinnamic or sinapinic acid in 50% acetonitrile/0.2% TFA was used as a MALDI matrix; 2 ll of sample (8 ng) and 2 ll of matrix solution were premixed in a tube, and 0.5 ll of the mixture was placed on the sample target and allowed to dry at ambient temperature. Positive-ion MALDI mass spectra were measured in reflectron mode on a Bruker BIFLEX II time-of-flight mass spectrometer (Bruker-Franzen, Bremen, Germany) equipped with a SCOUT 26 sample inlet, a gridless delayed extraction ion source, and a 337-nm nitrogen laser (Laser Science, Cambridge, MA). Spectra were accumulated from 20–50 laser shots. The instrument was calibrated externally using a mixture of peptide standards. Post-source decay (PSD) spectra were recorded in 12–16 segments, with each succeeding segment representing a 20% reduction in reflector voltage. About 50 shots were averaged per segment. Segments were pasted, calibrated, and smoothed under computer control by Bruker XMASS 5.0 software.

P. Halada et al. / Analytical Biochemistry 314 (2003) 235–242

ESI mass spectrometry Positive-ion mass spectra were recorded on an ion trap LCQDECA mass spectrometer (Finnigan, San Jose, CA) equipped with ESI ion source. Spray voltage was set at 5.5 kV and tube lens voltage was )10 V. The flow of the sheath gas (nitrogen 99.999%) was set at 55 arb units and the heated capillary was kept at 250 °C, with a potential of 32 V. Samples (4 ng/ll) dissolved in 50% acetonitrile were continuously infused into the ion source via linear syringe pump at a flow rate of 3 ll/min. Full-scan spectra were acquired over the m/z range 50– 2000 Da. A 0.1% solution of Ultramark 1621 (PCR, Inc., Gainesville, FL) in acetonitrile was used to calibrate the m/z scale of the instrument. For the MS2 and MS3 experiments, the activation amplitude for the parent ion was set to 25% and the CID spectra were recorded in the mass ranges 230–1400 and 95–360 Da, respectively. Edman degradation N-terminal amino acid sequence analysis was performed on an automated protein sequencer LF 3600D (Beckman Instruments, Inc., Fullerton, CA) according to the manufacturerÕs manual. NMR spectroscopy NMR spectra (400 MHz for 1 H, 100 MHz for 13 C) of the flavopeptide (2.3 mg) were recorded on a Varian INOVA-400 spectrometer (Varian, Inc., Palo Alto, CA) in D2 O at 30 °C. All 2D NMR experiments (gCOSY, LR COSY, TOCSY, ROESY, HMQC, HMBC) were performed using manufacturerÕs software. The sequence for 1D-TOCSY experiments [17] was obtained through Varian User Library. Chemical shifts were referenced to internal acetone ðdH 2.030, dC 30.50). 1 H NMR parameters were extracted from resolution-enhanced spectra or 1D-TOCSY; reported 13 C chemical shifts are HMQC or HMBC readouts.

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type of flavin–polypeptide linkage. This type of attachment was also indicated by intrinsic yellow–green fluorescence of the P2O band on SDS–PAGE gels under UV illumination (not shown). The stoichiometry of one flavin molecule per enzyme subunit was corroborated by spectrophotometry [15]. The major flavin-containing peptide generated from P2O by tryptic/chymotryptic digestion was purified using a two-step procedure based on anion exchange and reversed-phase chromatography (Fig. 1). The flavopeptide (5 mg) was obtained in the yield of 55% based on the FAD content (3.3 mg of FAD). Mass spectrometric analyses of the covalent flavin attachment site The MALDI-MS spectrum of the isolated intact flavopeptide exhibited an intense [M + H]þ ion peak at m/z 1313.4 (Fig. 2). Due to the unstable behavior of the FAD-containing peptide under MALDI conditions, fragments at m/z 966.3, 785.2, and 530.2 were also observed. The peaks were preliminarily attributed to the flavopeptide lacking AMP, FAD moiety, and deflavinylated peptide, respectively. These ions were generated during the desorption/ionization process in the ion source of the mass spectrometer and their formation allowed us to measure the corresponding fragments by PSD analysis and thus closely inspect the flavopeptide structure. The complementary series of fragments in the PSD spectrum of the ion m/z 785 clearly confirmed the presence of unmodified FAD cofactor (Fig. 2, inset). Fragment hþ (Fig. 3) with m/z 250 comprises adenine and ribose moiety, whereas ion ½j þ 2H þ at m/z 136 was

Results Isolation of P2O-derived flavopeptide P2O purified from mycelia of T. multicolor exhibited a single band on SDS–PAGE and had a subunit Mr of 67,329  135 Da based on linear-mode MALDI measurement of the intact protein. The enzyme showed characteristic flavoprotein absorption spectrum with maxima at 456, 345, and 275 nm in agreement with our previous data [15]. No flavin was released from the holoenzyme after denaturation with 5% trichloroacetic acid at 100 °C for 10 min, which suggested a covalent

Fig. 1. Elution profile of the tryptic/chymotryptic digest of P2O in the reversed-phase HPLC. Absorbance of the eluate was monitored at 220 nm (top) and 450 nm (bottom).

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Fig. 2. MALDI MS spectrum of the purified tryptic/chymotryptic peptide modified by FAD. The observed monoisotopic peaks correspond to [M + H]þ ions of FAD-containing peptide (1313.4), flavopeptide lacking AMP (966.3), and deflavinylated peptide (530.2). The ion m=z 785.2 comprises the whole FAD moiety as deduced from PSD analysis (inset). The complementary series of fragments clearly confirmed FAD structure. For the ion assignment see Fig. 3.

Fig. 3. Structure, atom numbering, mass spectrometric fragmentation, and diagnostic heteronuclear couplings of the flavopeptide isolated from tryptic/chymotryptic digest of pyranose 2-oxidase from Trametes multicolor. The fragment ion nomenclature was adopted from [18].

assigned to the adenine structure itself. Cleavage of the phosphodiester bond leads to an AMP fragment ½f þ 2H þ having m/z 348 and to its counterpart m/z 438 including ribitol and isoalloxazine ring. PSD analysis of the ion m/z 530 identified the amino acids present in the flavopeptide (Fig. 4).

Fig. 4. PSD mass spectrum of the unmodified peptide STHW. The sequence was unambiguously determined from the complete b- and ytype ion series. For the ion nomenclature see [19]. The numeric subscript n in a fragment ion name indicates the number of amino acid residues present in the given fragment. Immonium ions of individual amino acids are labeled with single letter code.

Immonium ions appearing in the low-mass region of the spectrum provided information about the amino acid composition, revealing histidine, serine, threonine, and tryptophan to be present in the peptide chain. Based on the complete series of N-terminal b- and C-terminal y-type ions resulting from the cleavage of a peptide bond, the sequence of the FAD peptide was then assigned as STHW. Considering the as yet single report of flavin link to threonine [11,12], His residue was proposed as the site of flavin attachment. This attachment was confirmed by PSD analysis of the ion m/z 966 of FAD-linked peptide lacking AMP (data not shown). Moreover, elemental compositions of selected diagnostic ions were measured with internal calibration under highresolution conditions (Table 1). To verify the results obtained by MALDI-MS, the FAD peptide was also analyzed on an ion-trap mass spectrometer equipped with electrospray ionization. The full-scan spectrum of the intact flavopeptide contained singly charged [M + H]þ and doubly charged [M+2H]2þ ions at m/z 1313.3 and 657.2, respectively, and ion species m/z 966.3 corresponding to the FAD-modified peptide lacking AMP. Under collisionally induced dissociation, the doubly charged precursor ion m/z 657.2 gave a complementary pair of fragments m/z 348 ½f þ 2H þ and 966 (Fig. 3). The former species lost ribosylphosphate in an MS3 experiment providing the ion ½j þ 2H þ at m/z 136

Table 1 MALDI mass spectrometric measurements of the diagnostic monoisotopic ions under high-resolution conditions Ion type

Elemental composition

[M + H]þ (calculated)

[M + H]þ (measured)

FAD-containing peptide Flavopeptide lacking AMP FAD Deflavinylated peptide

C51 H63 O22 N16 P2 C41 H49 O15 N11 P1 C27 H33 O15 N9 P2 C24 H32 O7 N7

1313.38 966.31 785.16 530.24

1313.37 966.30 785.17 530.24

P. Halada et al. / Analytical Biochemistry 314 (2003) 235–242

(not shown). These data strongly support the findings obtained by MALDI-MS. Taken together, mass spectrometric analyses of the flavopeptide isolated from the tryptic/chymotryptic digest of P2O from T. multicolor identified the sequence STHW with His residue as the covalent attachment site for FAD. The structure STXW was supported by N-terminal sequencing of the flavopeptide (Table 2). No PTH-amino acid derivative was detected in the third cycle, confirming the presence of a modified amino acid residue in this position. NMR identification of the flavin–peptide covalent link 1

H NMR spectrum measured in D2 O clearly indicated the presence of an 8a-modified flavin (one aromatic methyl, one low-field resonating methylene). COSY, TOCSY, and HMQC experiments revealed several spin systems: CH3 CH(O–)CHN– (Thr); –OCH2 CHN– (Ser); aromatic ABCD spin system + aromatic singlet + –CH2 CHN– (Trp); an AB system of two low-field resonating protons (8.705, 6.853 ppm, JAB ¼ 1:4 Hz) + –CH2 CHN– (His), –OCHOCH(O–)CH(O–) CH(O–)CH2 O– (ribose); –NCH2 CH(O–)CH(O–)CH (O–)CH2 O– (ribitol); two low-field singlets (adenine); and two aromatic singlets + aromatic methyl + methylene (isoalloxazine). LR COSY was used to link the –CH2 CHN– parts to their corresponding aromatic moieties in His and Trp residues. This method also provided the assignment of adenine H-8 based on its long-range coupling with ribose H-1. Chemical shifts and coupling constants of overlapped protons were extracted by 1D-TOCSY. The flavin system was identified by LR COSY, TOCSY, ROESY, and especially by HMBC. Nearly complete signal assignment (Table 3) was achieved by extensive use of all the above-mentioned 2D NMR methods. The agreement of 13 C chemical shifts with those published for FAD [20] was very good. Only C-2 and C-3 of ribose need to be interchanged on the basis of our results. There was also a reasonable similarity with the reported 1 H NMR spectra of histidyl [21] and imidazolyl [22] modified riboflavins. HMBC experiment provided cross-checks of the assignment and the assignments of most quaternary carbons in the molecule. The isoalloxazine moiety contained one methyl, one Table 2 Automated Edman degradation of the FAD-containing peptide isolated from the tryptic/chymotryptic digest of T. multicolor pyranose 2-oxidase Cycle

Amino acid residue

Recovery (pmol)

1 2 3 4

Ser Thr X Trp

93 86 n.o.a 18

a Not observed, blank cycle. No PTH-amino acid derivative extracted from the reaction cartridge.

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methylene, and two aromatic singlets. The aromatic proton exhibiting NOE to ribitol –NCH2 – was assigned to H-9, the other one was assigned to H-6. The flavin methyl showed NOE to H-6 and flavin methylene to H-9, indicating the 8a bonding type. In addition to the couplings to C-7, C-8, and C-9 of flavin, the 8a-methylene protons are coupled to both protonated histidine carbons (C-2 and C-4). Since their coupling partners would be C-2 and C-5 in the case of (N 1 -histidyl) link, the observed data are consistent with 8a-(N 3 -histidyl) attachment only (Fig. 3). Consequently, in P2O from T. multicolor, the 8amethyl group of FAD isoalloxazine ring is bound covalently through a tertiary amine linkage to histidine N 3 atom in the tetrapeptide STHW. The complete structure of the identified flavinylation region in T. multicolor P2O is shown in Fig. 3.

Discussion The structure elucidation of the P2O flavin-binding domain is of importance for understanding the enzyme reaction mechanisms and its optimized application. Mass spectrometry provided complete information on the peptide sequence around the flavin link, on the flavin type and its attachment site. To distinguish between 8a/ 7a- and N 3 /N 1 -histidylflavins, the nature of the chemical linkage of histidine to the methyl group of isoalloxazine ring was examined using NMR spectroscopy. The standard approach to the identification of a covalently linked flavoprotein [10,23] involves enzymatic degradation of isolated flavinylated peptide to its aminoacyl flavin central moiety, the physicochemical properties of which are compared with those of the known synthetic flavin derivatives. On the other hand, our protocol did not require the treatment of the flavopeptide with nucleotide pyrophosphatase, phosphatase, and aminopeptidase M; the intact flavopeptide was analyzed directly by MS and NMR. A potential of the presented approach is its ability to identify amino acids in the vicinity of the flavinylation site and to characterize possible modified flavin moieties and new types of cofactor attachment, which might be problematic using standard methods due to the lack of suitable standards for HPLC, fluorescence, and UV spectrometry. To the best of our knowledge, this is the first example of MS and NMR identification of the flavinylation region in a protein with still unknown sequence. The presented procedure does not replace in any case the well-established standard methods for analysis of covalent flavins [10,23]. Our method can serve as an alternative, which can be applied to identify a covalent flavinylation in a flavoprotein. The relatively high quantities of the purified flavopeptide (
2 mg) that we used for the analysis on our common NMR instrument

240

Table 3 NMR data of the FAD peptide (399.90 MHz for 1 H, 100.56 MHz for Residue Ser

His

Trp

IAOc

C@O a b C@O a b c C@O a b 2 4 5 C@O a b 2 3 3a 4 5 6 7 7a 2 4 6

dH —

4.037 3.783 —

4.148 3.847 0.866 —

4.443 3.034 2.932 8.705 6.853 —

4.129 2.481 6.635 — —

6.927 6.757 6.658 6.889 — — —

7.712

nH 0 1 2 0 1 1 3 0 1 1 1 1 1 0 0 1 2 1 0 0 1 1 1 1 0 0 0 1

M (J) t (5.1) d (5.1) d (4.9) dq (4.9,6.4) d (6.4) dd (8.6,5.6) dd (15.2,5.6) dd (15.2,8.6) d (1.4) d (1.4)

t (6.6) d (6.6)

m m m m

s

dC a 168.6 54.9 60.8 171.4 59.4 67.2 19.0 170.8 53.1 26.7

C, D2 O, 30 °C) HMBC H-a, H-b, H-a(Thr)

Residue c

IAO

H-a H-a, H-a(His) H-c H-a, H-c H-b, H-a(Trp) H-b Ribitol

136.0 120.5 130.2 174.7 53.9 27.0 124.4 108.9 126.5 111.7 122.2 119.4 118.2 135.7 n.o.b n.o. 132.9

H-4, H-8a H-2, H-8a, H-b H-2, H-4, H-b H-a

H-b H-2, H-2, H-6 H-6, H-4 H-5 H-2,

dH

nH

7 8 9 4a 5a 9a 10a 7-Me 8a

—

0 0 1 0 0 0 0 3 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 0 0 0 1

1 2 3 4 5

Ribose H-a, H-b H-5 H-7 Adenine H-4

Atom

1 2 3 4 5 2 4 5 6 8

—

7.408 — — — —

2.283 5.339 5.308 4.380 4.310 4.008 3.685 3.779 4.024 3.916 5.851 4.483 4.301 4.177 4.065 8.043 — — —

8.314

7-Me

dH , proton chemical shift; nH , number of protons; M, multiplicity; J , proton–proton coupling constants in Hz; dC , carbon chemical shift. a HMQC or HMBC readouts. b n.o., not observed. c IAO, isoalloxazine.

M (J)

s

s d (16.1) d (16.1) m m m m m m m d (5.2) dd (5.2,4.9) dd (4.9,4.0) m m s

s

dC a

HMBC

142.1 137.2 117.2 n.o. 135.7 132.1 n.o. 18.1 50.9

H-6, H-8a, 7-Me H-9, H-8a, 7-Me H-8a H-9 H-6 H-6 H-9

47.9 69.6 72.6 71.2 67.4 88.3 74.9 70.5 84.3 65.3 145.4 148.3 118.5 150.0 142.6

H-2, H-6 H-8 H-2 H-1(ribose)

P. Halada et al. / Analytical Biochemistry 314 (2003) 235–242

Thr

Atom

13

P. Halada et al. / Analytical Biochemistry 314 (2003) 235–242

could be significantly reduced by using higher magnetic fields and by introducing of (sub)microprobes [24,25] and cryogenic probes [26]; the last technique is very promising as it reduces the required sample amount to one fourth. Since the binding site of FAD was determined to be a histidine residue, the sequence around this flavin linkage residue was compared with sequence elements around His residues in the known sequences of P2O from the related fungi T. versicolor and T. hirsuta. The alignment of our sequence STHW with that around His129 and His140 , respectively, showed full agreement in both these enzymes. Accordingly, the site of covalent flavinylation occurs in the N-terminal area of the sequence and is located relatively close to the well-studied dinucleotide noncovalent binding motif [8] that was found near the N terminus of the P2O sequence [7]. The new sequence around the FAD attachment site in P2O from T. multicolor confirms, however, that the contiguous amino acids around the covalent flavin linkage residue in different flavoproteins bear little homology and show relatively poor conservation even for the same bonding type such as (N 3 -histidyl)-riboflavin. This further contributes to the opinion that covalent flavinylation is a self-catalytic process dependent on the primary folding of the polypeptide chain [9]. It is noticeable that the hydroxy amino acids Ser and Thr are frequently encountered in the close vicinity of His in the 8a-(N 3 ) linkage type [9] as we found also in the case of T. multicolor P2O. Moreover, the presence of the least bulky glycine residues near the FAD covalent attachment site in P2O from T. multicolor [7] is in good agreement with the majority of other covalent flavoproteins [9]. The covalent flavinylation is apparently preceded by the primary noncovalent capture of the cofactor. Two types of conserved domains for initial noncovalent FAD binding are known: the well-documented bab fold [8,27] and the FAD-binding fold of a recently revealed oxidoreductase family related to vanillyl-alcohol oxidase [28]. Some flavoenzymes exhibiting either type of the noncovalent binding can also incorporate covalently bound FAD cofactor. Based on our current and previous studies, pyranose 2-oxidase from T. multicolor can be classified as a covalent flavoprotein of the 8a-(N 3 histidyl)-FAD linkage type featuring the highly conserved motif Gly-X-Gly-X-X-Gly [7] that is a part of the bab fold domain for the noncovalent FAD binding. The molecular mechanisms underlying the transition between the two FAD-binding modes remain to be clarified.

Acknowledgments Financial support from the Grant Agency of the Czech Republic (206/99/1191) is gratefully acknowl-

241

edged. The work was also supported by Institutional Research Concept No. AV0Z5020903 and Grant 2002-9 (Program for Scientific-Technical Cooperation AKTION, Austria–Czech Republic).

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