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Originally published In Press as doi:10.1074/jbc.M506078200 on September 9, 2005

J. Biol. Chem., Vol. 280, Issue 46, 38831-38838, November 18, 2005
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Crystal Structure of Glucooligosaccharide Oxidase from Acremonium strictum

A NOVEL FLAVINYLATION OF 6-S-CYSTEINYL, 8{alpha}-N1-HISTIDYL FAD*

Chun-Hsiang Huang{ddagger}§1, Wen-Lin Lai§1, Meng-Hwan Lee§, Chun-Jung Chen||, Andrea Vasella**, Ying-Chieh Tsai§2, and Shwu-Huey Liaw{ddagger}{ddagger}{ddagger}3

From the {ddagger}Structural Biology Program, §Institute of Biochemistry, and Faculty of Life Science, National Yang-Ming University, Taipei 11221, Taiwan, the ||Biology Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan, the **Laboratorium für Organische Chemie, ETH Hönggerberg, CH-8093 Zürich, Switzerland, and the {ddagger}{ddagger}Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei 11217, Taiwan

Received for publication, June 3, 2005 , and in revised form, September 7, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Glucooligosaccharide oxidase from Acremonium strictum has been screened for potential applications in oligosaccharide acid production and alternative carbohydrate detection, because it catalyzes the oxidation of glucose, maltose, lactose, cellobiose and cello- and maltooligosaccharides. We report the crystal structures of the enzyme and of its complex with an inhibitor, 5-amino-5-deoxy- cellobiono-1,5-lactam at 1.55- and 1.98-Å resolution, respectively. Unexpectedly, the protein structure demonstrates the first known double attachment flavinylation, 6-S-cysteinyl, 8{alpha}-N1-histidyl FAD. The FAD cofactor is cross-linked to the enzyme via the C6 atom and the 8{alpha}-methyl group of the isoalloxazine ring with Cys130 and His70, respectively. This sugar oxidase possesses an open carbohydrate-binding groove, allowing the accommodation of higher oligosaccharides. The complex structure suggests that this enzyme may prefer a {beta}-D-glucosyl residue at the reducing end with the conserved Tyr429 acting as a general base to abstract the OH1 proton in concert with the H1 hydride transfer to the flavin N5. Finally, a detailed comparison illustrates the structural conservation as well as the divergence between this protein and its related flavoenzymes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Sugar oxidases and dehydrogenases that catalyze carbohydrate oxidation to the corresponding lactones are of considerable commercial importance as potential diagnostic reagents and industrial biocatalysts. For example, glucose oxidase (GOX)4 is widely used in analytical biochemistry and in the food industry (1). A search of the BRENDA enzyme data base (2) reveals that most of these enzymes are specific for a variety of mono- and disaccharides, and only a few enzymes are highly selective for oligosaccharides. These include galactose oxidase, cellobiose dehydrogenase (CDH), and glucooligosaccharide oxidase (GOOX).

GOOX from Acremonium strictum was screened with the aim of identifying enzymes with potential applications in oligosaccharide acid production and alternative carbohydrate assays (3). It is a monomeric glycoprotein with a covalently linked FAD and catalyzes the oxidization of a variety of carbohydrates with the concomitant reduction of molecular oxygen to hydrogen peroxide. A screening of more than 50 carbohydrates and derivatives showed that D-glucose, maltose, lactose, and cellobiose are good substrates. In addition, this enzyme can react with malto- and cellooligosaccharides, and hence the name of this novel oxidase. The broad substrate specificity of GOOX, particularly toward oligosaccharides, suggests that it may have great potential applicability.

To facilitate further characterization and potential industrial use of A. strictum GOOX, we have cloned the encoding gene, which is composed of a 25-residue signal peptide and a 474-residue mature protein (4). Although GOOX shows a similar substrate specificity as GOX, CDH, and pyranose oxidase (POX), it shares no sequence similarity with them. However, GOOX displays significant sequence homology to the FAD-binding domain (F domain) of berberine bridge enzyme-like proteins including three other sugar oxidases, a red alga hexose oxidase (CcHEOX), a tobacco nectar protein (nectarin V, NlNEC5), and a sunflower defense protein (HaCHOX) (5-7). Interestingly, although NlNEC5 catalyzes oxidation of glucose, and GOOX, CcHEOX and HaCHOX catalyze the oxidation of glucose, maltose, lactose, and cellobiose, the protein sequences of their substrate-binding domains (S domains) are quite diverse and apparently lack conserved carbohydrate-interacting residues. Moreover, structural and mutational studies demonstrated a consensus histidine for flavinylation in the F domain of berberine bridge enzyme-like proteins. Unexpectedly, mutation of the putative FAD-linking residue in GOOX, His70, to alanine, serine, cysteine, or tyrosine does not abolish the covalent FAD attachment (4). To gain structural insight into the FAD incorporation, substrate specificity, and catalytic mechanism, we have determined the GOOX structure in the absence, or presence of a product analog, 5-amino-5-deoxy-cellobiono-1,5-lactam (ABL).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Recombinant protein was expressed using the vector pPICZ{alpha}A in Pichia pastoris KM71, and then isolated by a Toyopearyl Phenyl-650 column with elution of a gradient of 2.5-0 M ammonium sulfate (4). Yellow protein crystals were grown in 25% polyethylene glycol monomethyl ether 550, 10 mM ZnSO4, and 0.1 M MES (pH 6.5), with a combination of 2 µl of reservoir solution and 2 µl of protein solution (35 mg/ml). Crystals appeared and reached their final dimensions in a month at 295 K. Only very few batches of recombinant proteins produce crystals of high quality. The high-resolution datasets were collected using the synchrotron radiation at the beamlines BL12B2 at SPring-8 (Harima, Japan) and NW12 at Photon Factory (Tsukuba, Japan). The data were processed using the HKL2000 program (8). The crystals belong to the P212121 space group with 1 monomer/asymmetric unit. Because of a lack of fresh crystals, the frozen native crystals were used for preparation of the ABL derivative by soaking crystals for 30 min in a reservoir solution containing 60 mM ABL. The x-ray data were collected on a RAXIS IV area detector with a Rigaku RU-300 rotating anode (MSC).

Heavy atom screening yielded one platinum derivative (K2PtI4) and sufficient phase information was derived from single-wavelength anomalous dispersion. Four Pt positions were identified with SHLEXD (9) and refined with SOLVE (10). The initial phase was improved significantly by direct method phasing refinement using OASIS (11) before density modification. An initial model with 248 polyalanine residues was built by RESOLVE (10). The electron density was improved greatly by fragment extension with OASIS, so that a complete model with side chains was built by ARP/wARP (12). The structure then underwent straightforward refinement against native data to 1.55 Å resolution using CNS (13). The structure of the ABL derivative was solved by molecular replacement using the refined coordinates mentioned above as the initial model. Statistics for data collection and refinement are summarized in TABLE ONE. More than 90% of the residues are in the most favored regions of Ramachandran plot, with the remaining in the additional allowed regions except for Thr307 and Arg445, because of hydrogen bond interactions. Crystals with better quality were grown in the presence of 10 mM ZnSO4, and four zinc ions bound to the enzyme were identified by the zinc anomalous data. Three zinc ions were replaced by platinum ions in the K2PtI4 derivative. Figs. 1, 3B, and 4A were generated by MolScript (14) and Raster3D (15), Figs. 2A and 3A by BobScript (14), Fig. 2B by LigPlot (16), and Fig. 3C by Grasp (17).


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  		TABLE ONE
Data collection and refinement statistics

Values in parentheses are for the highest resolution shell.

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The Overall Structure—The current model contains five linker residues and the 474-residue mature GOOX. The protein is composed of two distinct domains (Fig. 1). The F domain recruits the N-terminal residues 1-206 and the C-terminal residues 421-474 and folds into two ({alpha}+{beta}) subdomains. A small subdomain, residues 1-90, comprises a central four {beta}-strands ({beta}1-{beta}4) with the strand order 1, 2, 4, and 3, and with {beta}1 running antiparallel to the other strands. The {beta}-sheet is sandwiched by three helices ({alpha}A, {alpha}A' and {alpha}B), in which there is one disulfide bond between Cys6 of the {alpha}A helix and Cys55 of the {alpha}B helix. The second subdomain contains five antiparallel {beta}-strands ({beta}5-{beta}9) with the strand order 5, 6, 9, 7, and 8 and is surrounded by five {alpha}-helices ({alpha}C, {alpha}D, {alpha}D', {alpha}J, and {alpha}K). These two subdomains are packed against each other and accommodate the FAD cofactor between them.

Furthermore, the S domain (residues 207-420) is composed of a large seven-strand antiparallel {beta}-sheet ({beta}10-{beta}16) with the strand order 13, 10, 12, 11, 15, 16, and 14 and is flanked by five helices ({alpha}E-{alpha}I). This domain is positioned over the isoalloxazine ring of the FAD cofactor and constitutes most of the carbohydrate-binding groove. Two N-glycosylation modifications were detected in the S domain. The electron density showed clearly one ordered sugar residue each linked to Asn305 and Asn341, which was assigned as N-acetyl-D-glucosamine based solely on fitting to the electron density.

A Novel Flavinylation—Unexpectedly, the FAD cofactor is cross-linked to the enzyme at two attachment sites (Fig. 2A). One is the S{gamma} atom of Cys130 bound to the C6 atom of the isoalloxazine ring, whereas the other is the N{delta}1 atom of His70 bound to the 8{alpha}-methyl group (6-S-cysteinyl, 8{alpha}-N1-histidyl FAD). The ADP-ribityl moiety is embedded in the F domain and is completely solvent-inaccessible. The adenine and ribose groups form eight hydrogen bonds with two water molecules and the backbone atoms of Ala65, Gly106, and Val195 (Fig. 2B). The negatively charged pyrophosphate group interacts with a constellation of backbone NH groups of Gly67, Gly68, Gly69, His70, Ser71, Gly134, and Gly137. In addition, the ribityl group makes four hydrogen bonds with one water molecule, the backbone of His70, and the side chains of His138 and Asn. The isoalloxazine ring lies at the juncture of the F and S domains. In addition to the covalent linking to His70 and Cys130, the ring forms five hydrogen bonds with two water molecules, the backbones of Thr129 and Tyr144, and the side chain of Tyr426.



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  		FIGURE 1.
Stereo view of the GOOX structure. The protein consists of a FAD-binding (F) and a substrate-binding (S) domain, colored in red (helix) and green (strand). The intermediate analogue ABL (magenta), the cofactor FAD (black), the linking residues His70 and Cys130, and the glycosylated Asn305 and Asn341 are displayed as ball-and-stick representations. The two major FAD-interacting segments are highlighted in cyan. The hydrogen bonds between the backbone of one residue at the N terminus of the {beta}9 strand (Val195 in GOOX) with the adenine N1 and N6 are strictly conserved.

 
Five types of covalent flavinylation have been identified up to the present, and flavinylation has been shown to be an autocatalytic process (18). The four types of flavinylation at the 8{alpha}-methyl group cross-linking to His (N{delta}1 and N{epsilon}2), Tyr (O{eta}), and Cys (S{gamma}), were shown to be involved in modulation of the redox potential of the isoalloxazine ring. Notably and in contradistinction, an unusual 6-S-cysteinyl modification was only observed in the FMN cofactor of trimethylamine dehydrogenase and its homologues, dimethylamine dehydrogenase and histamine dehydrogenase (19, 20). This unusual 6-S-cysteinyl attachment has been suggested to prevent enzyme inactivation by hydroxylation at the C6 atom. Thus, GOOX possesses a novel form of covalent flavinylation; it is the first example of 6-S-cysteinyl FAD and the first double covalent linkage identified to date. Replacement of His70 with alanine, serine, cysteine, or tyrosine decreased the kcat value 50-600-fold but had little effect on the Km (4). These four His70 mutants still contain a covalently linked FAD. These indicate that the covalent attachment via the 8{alpha}-methyl group with His70 is able to enhance the oxidative power of the flavin, as has been shown in other covalent flavoenzymes (18). Nevertheless, the functional role of the 6-S-cysteinyl flavinylation in GOOX is still under investigation.

Substrate Specificity—ABL resembles cellobionolactone, the oxidation product of cellobiose, with replacement of the endocyclic O5 with an NH group (Fig. 3). This replacement suppresses the spontaneous hydrolysis of ABL to cellobionic acid. ABL is the only inhibitor against GOOX activity found to date. Approximately 50% of glucose oxidation activity was lost in the presence of 25 mM ABL. In addition, ABL has been shown to be an inhibitor for CDH. The structure of CDH in complex with this inhibitor has been solved and used to delineate a reaction mechanism for CDH (21). In the complex structure presented here, ABL is identified by its strong electron density that correlates well with the shape of D-glucopyranosyl rings, and is firmly embedded on the si face of the isoalloxazine ring (Fig. 3A). The lactam moiety of ABL is bound in the -1 subsite. The lactam C1 atom, which corresponds to the site of oxidation in cellobiose, binds in front of the flavin N5 with a distance of 3.35 Å and an angle with the N5-N10 atoms of 105°. It should be noted that like for the CDH complex (21), the lactam C1 and O1 atoms are almost perfectly aligned with the flavin N5 and C4{alpha}, respectively.

The lactam O1 atom interacts with Tyr429O{eta} (2.78 Å) and Gln384N{epsilon}2 (3.14 Å), and the endocyclic NH makes close contacts with the Tyr72O{eta} (3.34 Å) (Fig. 3B). The equatorial OH2 group forms hydrogen bonds with Thr129O{gamma}1 (2.84 Å), Arg245N{eta}1 (3.13 Å), and the isoalloxazine O4 (3.23 Å), the equatorial OH3 with Arg245N{eta}2 (3.15 Å) and Gln353O{epsilon}1 (2.86 Å), and the OH6 with Tyr72O{eta} (2.88 Å) and Tyr386O{eta} (3.67 Å). Thus, a total of nine hydrogen bonds are formed at the -1 subsite. In contrast, the -2 glucosyl moiety of ABL forms only one direct protein-carbohydrate hydrogen bond between OH6 and Gln353O{epsilon}1 (3.30 Å). Additionally, the OH2 and OH6 groups form two and three water-mediated hydrogen bonds, respectively. Tyr300 and Trp351 stack on the pyranose ring with an interplanar distance of 4.2-4.3 Å. This type of stacking interaction is very common in protein-carbohydrate recognition. The ABL binding does not induce any significant conformational change except for the side chain of Glu247.

On the basis of the bound ABL molecule described above, a variety of carbohydrate molecules were modeled manually in the carbohydrate-binding groove, and the model was subjected to energy minimization with CNS. D-Glucose is the only monosaccharide substrate for GOOX (3). Simulation of the complexes shows that other hexoses and derivatives form either fewer hydrogen bonds or unfavorable contacts with the residues surrounding the substrate-binding groove. The axial OH3 group in allose and the absence of an OH2 in 2-deoxy-D-glucose and of the exocyclic CH2OH in D-xylose would entail a small number of hydrogen bonds. The axial OH2 in mannose, the axial OH4 in galactose, and the equatorial NH2 in glucosamine would cause unfavorable contacts with Gln353, Trp351, and Arg245, respectively. Modification at the C1, C2, and C6 positions such as phosphorylation and N-acetylation would cause steric hindrance because of lack of appropriate space. Like CDH and galactose oxidase (21, 22), GOOX possesses an open carbohydrate-binding groove, so that the non-reducing end of the glucose residue can stick out into the solvent and be exposed on the protein surface (Fig. 3C). This explains why the enzyme is able to utilize oligosaccharides as good substrates (4). In contradistinction, the crystal structures of GOX and POX have revealed a "size-exclusion mechanism," in which the shape of the active site cavity is such that the enzyme can only accept monosaccharides (23, 24).



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  		FIGURE 2.
The FAD-binding site. A, stereo view of the 2Fo - Fc electron density map for the FAD cofactor and the covalently bound His70 and Cys130 contoured at 2{sigma} level. The density map demonstrates the first known double attachment flavinylation, 6-S-cysteinyl, 8{alpha}-N1-histidyl FAD. B, schematic diagram of GOOX interactions with the FAD cofactor. The residues forming hydrogen bonds to FAD are shown in ball-and-stick representation. Hydrogen bonds are presented as dashed lines and the interatomic distances are shown in angstroms. "Radiating" spheres indicate hydrophobic contacts between the cofactor and the surrounding residues.

 



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  		FIGURE 3.
The carbohydrate-binding site. A, stereo view of the 2Fo - Fc electron density map for the intermediate analogue ABL contoured at 1.2{sigma} level. B, stereo view of the interaction networks between ABL and GOOX. Hydrogen bonds are shown as green dashed lines. There are eight direct hydrogen bonds between the protein and the -1 sugar, but only one hydrogen bond between the protein and the -2 sugar (see "Results and Discussion" for a detailed explanation). C, molecular surfaces of GOOX. The protein surface is colored for electrostatic potential from -20 kBT (red) to 20 kBT (blue), reflecting its pI value of 4.3-4.5 (3). The FAD cofactor is colored in black, and the modeled cellohexaose is in green. The open carbohydrate-binding groove explains why oligosaccharides are good substrates.

 
Catalytic Mechanism—Most of the FAD-dependent oxidases and dehydrogenases are proposed to function via a hydride transfer rather than by a carbanion mechanism (25, 26). The pH optimum of 10 for GOOX implies that a tyrosine residue may serve as a general base (3). Based on the ABL binding, the substrate cellobiose was modeled into the active site. The reducing end C1 atom of the -1 sugar binds in front of the flavin N5 at a distance of 2.86 Å and an angle with the N5-N10 atoms of 113°. These values are in agreement with those typically observed for flavoenzymes, and appropriate for a direct hydride transfer (25, 26). However, no appropriate general base for carbanion formation can be identified. The hemiacetal OH1 group of the {beta}-anomer at the -1 subsite could interact with Tyr429O{eta}1 (3.11 Å), Gln384N{epsilon}2 (3.69 Å), and the isoalloxazine O4 (3.11 Å), whereas that of the {alpha} anomer may make close contacts with Thr129O{gamma} (3.17 Å) and the isoalloxazine O4 (3.25 Å). The distance between the reducing end H1 of the {beta}-anomer and the flavin N5 is 2.13 Å, whereas that for C1H of the {alpha} to the flavin N5 is 2.64 Å. Therefore, GOOX may preferentially oxidize the {beta}-anomer possessing an equatorial hydroxyl group, with the conserved Tyr429 acting as a general base.



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  		SCHEME 1.
The proposed catalytic mechanism for GOOX.

 
As is common for flavoenzymes, the reaction mechanism of GOOX consists of two half-reactions (Scheme 1). The reductive half-reaction is involved in the oxidation of the free reducing end {beta}-D-glucosyl residue to glucono-1,5-lactone by hydride transfer to the N5 atom, probably initiated by proton abstraction from the OH1 group by Tyr429. The lactone product is spontaneously hydrolyzed to gluconic acid. In the oxidative half-reaction, regeneration of the oxidized FAD by molecular oxygen yields hydrogen peroxide. Water-mediated hydrogen bonds between Asp355 and Tyr429 suggest that Asp355 may assist in proton transfer by lowering the pKa value of Tyr429 through a water molecule. Interactions between Gln384 and the reducing end OH1 group suggest that Gln384 may contribute to position the substrate and facilitate proton abstraction by Tyr429. His138, Tyr426, and the backbone NH of Tyr144 may be involved in stabilization of the anionic form of the reduced flavin. A large water-filled channel above the carbohydrate-binding groove and toward the flavin moiety may act as an entry point for the second substrate, molecular oxygen (Fig. 3C).

Structural Conservation and Divergence in the PCMH Superfamily—A structural homology search by DALI (27) revealed that GOOX displays significant structural similarity to Zea mays cytokinin dehydrogenase (ZmCKX), Brevibacterium sterolicum cholesterol oxidase 2 (BsCOX2), Pseudomonas putida p-cresol methylhydroxylase (PpPCMH), Penicillium simplicissimum vanillyl-alcohol oxidase (PsVAO), and Escherichia coli D-lactate dehydrogenase (EcDLDH), with root mean square deviations of 2.8 Å (414 C{alpha} atoms with 16% sequence identity), 3.5 Å (398 C{alpha} atoms with 10% identity), 3.5 Å (398 C{alpha} atoms with 16% identity), 3.4 Å (404 C{alpha} atoms with 13% sequence identity), and 3.6 Å (369 C{alpha} atoms with 12% identity), respectively (28-32). The structural homologies of these flavoproteins seem to suggest a divergent evolutionary relation, and thereby are defined as the PCMH superfamily because the crystal structure of PpPCMH was the first one reported (33). However, the relative orientation between the F and S domains does vary greatly across the different structures. The F domain also shares structural homology to E. coli uridine diphospho-N-acetylenolpyruvylglucosamine reductase (EcMurB), Oligotropha carboxidovorans CO dehydrogenase (OcCODH), and Bos taurus xanthine oxidase (BtXOX) (34-36).

The FAD binding modes are one of the least conserved properties among flavoproteins, and the flavinylation sites are often located in poorly conserved regions (18, 37). Although these PCMH members share a similar F domain, they contain different FAD binding types. In addition to the novel flavinylation in GOOX (6-S-cysteinyl, 8{alpha}-N1-histidyl FAD), the FAD cofactor utilizes its 8{alpha}-methyl group cross-linking to His105 in ZmCKX and His121 in BsCOX2 (8{alpha}-N1-histidyl FAD), to His422 in PsVAO (8{alpha}-N3-histidyl FAD), and to Tyr384 in PpPCMH (8{alpha}-O-tyrosyl FAD). The remaining members bind to FAD non-covalently.

The F domain is much more superimposable than the S domain, perhaps because of distinct substrate binding but a similar FAD recognition mechanism across the enzymes. The eight {beta}-strands ({beta}2-{beta}9, 60 structurally equivalent residues) of the F domain, overlay within 0.8-1.4 Å root mean square deviations (Fig. 4A). Interestingly, these members utilize many main-chain atoms for the FAD binding, and hence the sequences of the FAD-interacting residues are divergent, whereas their spatial positions are convergent in four regions (Figs. 1 and 4B). The first region is located in the loop between the {beta}3 and {beta}4 strands and shares similar features with the "P-loop," which is highly conserved in nearly all nucleotide-binding proteins and responsible for interaction with the pyrophosphate group of the cofactor NDP moiety (38). The second region involves the short {alpha}D helix, and the third region is at located the N terminus of the {beta}9 strand. It is worth noting that the hydrogen bonds between the adenine N1 and N6 and the backbone of one residue at the N terminus of the {beta}9 strand (Val195 in GOOX) are strictly conserved, even in EcMurB, OcCODH, and BtXOX. The last region is at the C-terminal tail, and the residues utilize the side chains for the cofactor binding.



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  		FIGURE 4.
Structural conservation and divergence in the PCMH superfamily. A, stereo view of structural superposition of the {beta}2-{beta}9 strands of GOOX (red), ZmCKX (green), BsCOX2 (blue), PpPCMH (black), PsVAO (yellow), EcDLDH (cyan), and EcMurB (pink). The {beta}-strands correspond closely, whereas the {alpha}-helices and surface-exposed loops diverge significantly. B, structure-based sequence alignment. The accession codes are listed in the right column. The secondary structure elements for GOOX are labeled (ss). The number of residues present in gaps is indicated in parentheses. The flavinylation sites are colored in red and indicated by *. Residues making close contacts with FAD are shaded in magenta and those for the conservative hydrophobic core are in yellow. The substrate-binding residues are shaded in cyan, and the putative conserved sugar-binding residues in GOOX, NlNEC5, HaCHOX, and CcHEOX are in green. The diverse S domains of these sugar oxidases were aligned on the basis of the secondary structure prediction and the conservative hydrophobic core.

 
In contrast, the seven {beta}-strands ({beta}10-{beta}16) of the S domain, particularly the {beta}11 and {beta}14-{beta}16 stands, varies greatly across the different structures. These diverse four {beta}-strands make up the majority of the substrate-binding site (Figs. 1 and 4B) and result in the highly divergent architectures of the substrate-binding site needed for the various different substrates ranging from a small lactate to the bulky cholesterol and long chain oligosaccharides. The closed conformations of ZmCKX, BsCOX2, PpPCMH, and PsVAO, limit the size of the substrate-binding pocket and thus result in a "size-exclusion mechanism" (28-31). In contradistinction, GOOX has an open carbohydrate-binding groove to accommodate larger oligosaccharides.

Convergent and Divergent Evolution in FAD-utilizing Sugar Oxidases—According to the structural fold, flavoenzymes have been classified into many superfamilies (37). Interestingly, folding topology does not correlate with enzyme function. For example, GOOX and BsCOX2 belong to the PCMH superfamily, whereas GOX, POX, CDH, and BsCOX1 belong to the glutathione reductase superfamily. Location of the active center at the re face of the isoalloxazine ring is generally conserved within the glutathione reductase superfamily, whereas that of the PCMH members is on the si side. Therefore, these structures provide elegant examples of convergent evolution, where starting from different ancestral folds, the same FAD-assisted glucose (or cholesterol) oxidation is achieved through opposite flavin faces within distinct substrate-binding sites. In addition, members of the PCMH superfamily have a remarkable tendency toward covalent flavinylation at the 8{alpha}-methyl group even for different types of flavinylation (18), whereas those of the glutathione reductase superfamily prefer a non-covalent binding mode. This may imply that the former FAD-binding fold facilitates flavinylation to optimize the active site for enhancement of its oxidative power.

Interestingly, even starting from a similar structural fold, sugar oxidases evolve some dissimilar residues for interaction with the common substrates. Within the glutathione reductase superfamily, GOX, POX, and CDH catalyze the oxidation of glucose, and share a similar F domain; however, their S domains lack conserved carbohydrate-interacting residues except for the conserved histidine serving as a general base (21, 23, 24). Similarly, within the PCMH superfamily, although GOOX resembles NlNEC5, CcHEOX, and HaCHOX in substrate specificity and F domain, the protein sequences of their S domains are quite diverse (Fig. 4B). The sugar-binding resides Tyr72, Arg245, Gln384, and Tyr429 in GOOX, all at the -1 subsite, may also be conserved in NlNEC5, CcHEOX, and HaCHOX. However, the other residues including Thr129, Tyr300, Trp351, and Gln353, apparently are not conserved. Therefore, these sugar oxidases have evolved a similar FAD-assisted oxidation mechanism but different substrate recognition, resulting in distinct binding affinities to the mono- and disaccharides, with Km values ranging from 50 to 30 mM.


   FOOTNOTES
 
The atomic coordinates and structure factors (codes 1ZR6 and 2AXR) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by the National Science Council (NSC93-2311-B010-009). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence may be addressed: Institute of Biochemistry, National Yang-Ming University, Taipei 11221, Taiwan. Tel.: 886-2-2826-7125; Fax: 886-2826-4843; E-mail: tasiyc{at}edu.tw.

3 To whom correspondence may be addressed: Faculty of Life Science, National Yang-Ming University, Taipei 11221, Taiwan. Tel.: 886-2-2826-7278; Fax: 886-2-2820-2449; E-mail: shliaw{at}ym.edu.tw.

4 The abbreviations used are: GOX, glucose oxidase; GOOX, glucooligosaccharide oxidase; POX, pyranose oxidase; CDH, cellobiose dehydrogenase; F domain, FAD-binding domain; S domain, substrate-binding domain; ABL, 5-amino-5-deoxy-cellobiono-1,5-lactam; NlNEC5, nectarin V from Nicotiana langsdorffii; CcHEOX, a hexose oxidase from Chondrus crispus; HaCHOX, a defense protein from Helianthus annuus with carbohydrate oxidase activity; ZmCKX, Zea mays cytokinin dehydrogenase; BsCOX2, Brevibacterium sterolicum cholesterol oxidase 2; PpPCMH, Pseudomonas putida p-cresol methylhydroxylase; PsVAO, Penicillium simplicissimum vanillyl-alcohol oxidase; EcDLDH, E. coli D-lactate dehydrogenase; EcMurB, E. coli uridine diphospho-N-acetylenolpyruvylglucosamine reductase; OcCODH, Oligotropha carboxidovorans CO dehydrogenase; BtXOX, Bos taurus xanthine oxidase; MES, 4-morpholineethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank Dr. H. F. Fan and J. W. Wang for kind assistance in structural determination using OASIS.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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