Crystal Structure of Glucooligosaccharide Oxidase from Acremonium strictum

Abstract

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, 8α-N1-histidyl FAD. The FAD cofactor is cross-linked to the enzyme via the C6 atom and the 8α-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 β-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. 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, 8α-N1-histidyl FAD. The FAD cofactor is cross-linked to the enzyme via the C6 atom and the 8α-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 β-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. 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) 4The 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. is widely used in analytical biochemistry and in the food industry (1.Meyer A.S. Isaksen A. Trends Food Sci. Technol. 1995; 6: 300-304Crossref Scopus (142) Google Scholar). A search of the BRENDA enzyme data base (2.Schomburg I. Chang A. Ebeling C. Gremse M. Heldt C. Huhn G. Schomburg D. Nucleic Acids Res. 2004; 32: 431-433Crossref PubMed Google Scholar) 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.Lin S.F. Yang T.Y. Inukai T. Yamasaki M. Tsai Y.C. Biochim. Biophys. Acta. 1991; 1118: 41-47Crossref PubMed Scopus (56) Google Scholar). 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.Lee, M. H., Lai, W. L., Lin, S. F., Hsu, C. S., Liaw, S. H., and Tsai, Y. C. (2005) Appl. Environ. Microbiol. in pressGoogle Scholar). 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.Groen B.W. De Vries S. Duine J.A. Eur. J. Biochem. 1997; 244: 858-861Crossref PubMed Scopus (22) Google Scholar, 6.Carter C.J. Thornburg R.W. Plant Physiol. 2004; 134: 460-469Crossref PubMed Scopus (86) Google Scholar, 7.Custers J.H. Harrison S.J. Sela-Buurlage M.B. van Deventer E. Lageweg W. Howe P.W. van der Meijs P.J. Ponstein A.S. Simons B.H. Melchers L.S. Stuiver M.H. Plant J. 2004; 39: 147-160Crossref PubMed Scopus (89) Google Scholar). 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.Lee, M. H., Lai, W. L., Lin, S. F., Hsu, C. S., Liaw, S. H., and Tsai, Y. C. (2005) Appl. Environ. Microbiol. in pressGoogle Scholar). 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). Recombinant protein was expressed using the vector pPICZαA 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.Lee, M. H., Lai, W. L., Lin, S. F., Hsu, C. S., Liaw, S. H., and Tsai, Y. C. (2005) Appl. Environ. Microbiol. in pressGoogle Scholar). 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.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar). 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.Schneider T.R. Sheldrick G.M. Acta Crystallogr. Sect. D. 2002; 58: 1772-1779Crossref PubMed Scopus (1578) Google Scholar) and refined with SOLVE (10.Terwilliger T.C. Methods Enzymol. 2003; 374: 22-37Crossref PubMed Scopus (433) Google Scholar). The initial phase was improved significantly by direct method phasing refinement using OASIS (11.Wang J.W. Chen J.R. Gu Y.X. Zheng C.D. Jiang F. Fan H.F. Terwilliger T.C. Hao Q. Acta Crystallogr. Sect. D. 2004; 60: 1244-1253Crossref PubMed Scopus (32) Google Scholar) before density modification. An initial model with 248 polyalanine residues was built by RESOLVE (10.Terwilliger T.C. Methods Enzymol. 2003; 374: 22-37Crossref PubMed Scopus (433) Google Scholar). 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.Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2564) Google Scholar). The structure then underwent straightforward refinement against native data to 1.55 Å resolution using CNS (13.Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar). 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.Esnouf R.M. Acta Crystallogr. Sect. D. 1999; 55: 938-940Crossref PubMed Scopus (850) Google Scholar) and Raster3D (15.Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3875) Google Scholar), Figs. 2A and 3A by BobScript (14.Esnouf R.M. Acta Crystallogr. Sect. D. 1999; 55: 938-940Crossref PubMed Scopus (850) Google Scholar), Fig. 2B by LigPlot (16.Wallace A.C. Laskowski R.A. Thornton J.M. Protein Eng. 1995; 8: 127-134Crossref PubMed Scopus (4368) Google Scholar), and Fig. 3C by Grasp (17.Nicholls A. Bharadwaj R. Honig B. Biophys. J. 1993; 64: A166Google Scholar).TABLE ONEData collection and refinement statisticsNativeK2PtI4ABLData collectionUnit cell (Å)53.1, 90.9, 111.753.8, 91.5, 113.053.7, 91.1, 112.3Wavelength (Å)0.71.07181.5418Resolution range (Å)50-1.55 (1.58-1.55)50-2.0 (2.07-2.0)50-1.98 (2.05-1.98)Total observations786,463 (27,250)486,389 (40,996)124,905 (7,832)Unique reflections77,613 (4,395)37,715 (3,628)35,571 (3,144)Completeness (%)98.1 (95.9)97.8 (95.6)90.5 (81.8)I/σ 〈I 〉30.1 (3.3)25.8 (9.2)15.0 (3.5)Rmerge(%)6.1 (27.0)5.1 (14.3)8.9 (29.2)RefinementResolution range (Å)50-1.55 (1.56-1.55)50-1.98 (1.99-1.98)Reflections (F > 0 σF)77,613 (1,471)35,571 (628)Rcryst (%) for 90% data18.1 (24.8)20.0 (28.4)Rfree (%) for 10% data20.0 (25.4)24.0 (30.2)Root mean Protein Protein FAD FAD ABL in a carbohydrate-binding of the electron density for the ABL at of the between ABL and are as are direct hydrogen between the protein and the only one hydrogen bond between the protein and the sugar and for a detailed molecular of The protein is for potential from to its of (3.Lin S.F. Yang T.Y. Inukai T. Yamasaki M. Tsai Y.C. Biochim. Biophys. Acta. 1991; 1118: 41-47Crossref PubMed Scopus (56) Google Scholar). The FAD cofactor is in and the is in The open carbohydrate-binding are good catalytic for FAD-binding of the electron density for the FAD cofactor and the covalently bound and Cys130 at The density demonstrates the first known double attachment flavinylation, 6-S-cysteinyl, 8α-N1-histidyl FAD. of GOOX with the FAD The residues hydrogen to FAD are in are as and the are in between the cofactor and the The model residues and the 474-residue mature The protein is composed of domains The F domain the residues and the residues and into A residues a four with the 1, and and with to the other The is by three and in which is one bond between of the and of the The with the and and is by and These are against other and the FAD cofactor between them. the S domain is composed of a with the and and is by This domain is the isoalloxazine ring of the FAD cofactor and most of the carbohydrate-binding were in the S The electron density showed one sugar residue linked to and which was as on to the electron A the FAD cofactor is cross-linked to the enzyme at attachment is the atom of Cys130 bound to the C6 atom of the isoalloxazine the other is the atom of bound to the 8α-methyl group 8α-N1-histidyl The is in the F domain and is The and hydrogen with and the of and The group with a of of His70, and In addition, the group four hydrogen with one the of His70, and the side chains of and The isoalloxazine ring at the of the F and S In to the covalent to and the ring hydrogen with the of and and the side of of covalent flavinylation have been identified to the and flavinylation has been to an M. N.S. Protein Sci. 1998; PubMed Scopus Google Scholar). The four of flavinylation at the 8α-methyl group to and and were to in of the potential of the isoalloxazine and in an was only in the cofactor of dehydrogenase and its dehydrogenase and dehydrogenase M. J. R. N.S. 1997; PubMed Scopus Google Scholar, A. K. T. 2004; PubMed Scopus Google Scholar). This attachment has been to enzyme by at the C6 GOOX possesses a novel of covalent it is the first of FAD and the first double covalent identified to of with alanine, serine, cysteine, or tyrosine the on the (4.Lee, M. H., Lai, W. L., Lin, S. F., Hsu, C. S., Liaw, S. H., and Tsai, Y. C. (2005) Appl. Environ. Microbiol. in pressGoogle Scholar). These four a covalently linked FAD. These that the covalent attachment via the 8α-methyl group with is to the of the as has been in other covalent M. N.S. Protein Sci. 1998; PubMed Scopus Google Scholar). the of the flavinylation in GOOX is the oxidation product of cellobiose, with replacement of the with an group This replacement the of ABL to acid. ABL is the only against GOOX to of glucose oxidation was in the presence of mm ABL. In addition, ABL has been to an for The structure of in complex with this has been solved and used to a for G. G. A. C. J. Biol. 2003; PubMed Scopus Google Scholar). In the complex structure ABL is identified by its electron density that well with the of and is on the of the isoalloxazine ring The of ABL is bound in the The which to the of oxidation in cellobiose, in of the flavin with a of Å and an with the of It that for the complex G. G. A. C. J. Biol. 2003; PubMed Scopus Google Scholar), the and are with the flavin and respectively. The atom with and and the with the The group hydrogen with and the isoalloxazine the with and and the with and a of hydrogen are at the In the of ABL only one direct hydrogen bond between and the and and three hydrogen respectively. and on the pyranose ring with an of This of is very in The ABL does not significant except for the side of the of the bound ABL a variety of carbohydrate were in the carbohydrate-binding groove, and the model was to with is the only substrate for GOOX (3.Lin S.F. Yang T.Y. Inukai T. Yamasaki M. Tsai Y.C. Biochim. Biophys. Acta. 1991; 1118: 41-47Crossref PubMed Scopus (56) Google Scholar). of the shows that other and derivatives hydrogen or with the residues the substrate-binding The group in and the of an in and of the in a of hydrogen The in the in and the in with and respectively. at the and C6 positions as and because of lack of and galactose oxidase G. G. A. C. J. Biol. 2003; PubMed Scopus Google Scholar, S.J. Sci. S. A. PubMed Scopus Google Scholar), GOOX possesses an open carbohydrate-binding groove, so that the end of the glucose residue can into the and on the protein This the enzyme is to as good (4.Lee, M. H., Lai, W. L., Lin, S. F., Hsu, C. S., Liaw, S. H., and Tsai, Y. C. (2005) Appl. Environ. Microbiol. in pressGoogle Scholar). In the crystal structures of and have a in which the of the is that the enzyme can only G. S. J. Schomburg D. Acta Crystallogr. Sect. D. 1999; 55: PubMed Scopus Google Scholar, C. D. C. J. Biol. 2004; PubMed Scopus Google Scholar). of the oxidases and dehydrogenases are to via a hydride transfer than by a A. Trends Biochem. Sci. PubMed Scopus Google Scholar, 2004; 32: PubMed Scopus Google Scholar). The of 10 for GOOX that a tyrosine residue may as a general base (3.Lin S.F. Yang T.Y. Inukai T. Yamasaki M. Tsai Y.C. Biochim. Biophys. Acta. 1991; 1118: 41-47Crossref PubMed Scopus (56) Google Scholar). on the ABL the substrate cellobiose was into the The reducing end atom of the sugar in of the flavin at a of Å and an with the of These are in with for and for a direct hydride transfer A. Trends Biochem. Sci. PubMed Scopus Google Scholar, 2004; 32: PubMed Scopus Google Scholar). However, no general base for can The OH1 group of the at the with and the isoalloxazine that of the may with and the isoalloxazine The between the reducing end H1 of the and the flavin is that for of the to the flavin is GOOX may the an with the conserved Tyr429 acting as a general is for the of GOOX of The is in the oxidation of the reducing end β-d-glucosyl residue to by hydride transfer to the by proton from the OH1 group by The product is to acid. In the of the FAD by molecular oxygen hydrogen peroxide. hydrogen between and Tyr429 that may in proton transfer by the of Tyr429 a between and the reducing end OH1 group that may to the substrate and facilitate proton by and the of may in of the of the A above the carbohydrate-binding and toward the flavin may as an for the molecular oxygen and in the structural homology search by S. J. C. A. M. Nucleic Acids Res. PubMed Scopus Google Scholar) that GOOX displays significant structural similarity to Zea mays cytokinin dehydrogenase Brevibacterium sterolicum cholesterol oxidase 2 Pseudomonas putida p-cresol Penicillium simplicissimum vanillyl-alcohol oxidase and coli d-lactate dehydrogenase with mean of Å with sequence Å with 10% Å with Å with sequence and Å with E. A. A. J. Biol. 2004; PubMed Scopus Google Scholar, A. J. Biol. 2003; PubMed Scopus Google Scholar, L.M. Chen PubMed Scopus Google Scholar, van van A. J. Biol. PubMed Scopus Google Scholar, E.A. C. D. Sci. S. A. PubMed Scopus Google Scholar). The structural of these to a and are as the because the crystal structure of was the first one Chen 1991; Scopus Google Scholar). However, the between the F and S domains does greatly the The F domain shares structural homology to E. coli uridine diphospho-N-acetylenolpyruvylglucosamine Oligotropha carboxidovorans CO dehydrogenase and Bos taurus xanthine oxidase J.M. 1997; PubMed Scopus Google Scholar, R. R. Sci. S. A. 2002; PubMed Scopus Google Scholar, C. K. T. T. Sci. S. A. PubMed Scopus Google Scholar). The FAD are one of the conserved and the flavinylation are in conserved regions M. N.S. Protein Sci. 1998; PubMed Scopus Google Scholar, D. Protein Sci. PubMed Scopus Google Scholar). Although these a similar F domain, FAD In to the novel flavinylation in GOOX 8α-N1-histidyl the FAD cofactor its 8α-methyl group to in and in to in and to in The remaining to FAD The F domain is more than the S domain, because of substrate a similar FAD the The 60 of the F domain, Å mean Interestingly, these for the FAD and hence the sequences of the residues are their positions are in four regions 1 and The first is in the between the and and shares similar with the which is highly conserved in proteins and for with the group of the cofactor L.M. Biol. 2004; PubMed Google Scholar). The the and the is at the of the It is that the hydrogen between the and and the of one residue at the of the in are in EcMurB, OcCODH, and The is at the and the residues the side chains for the cofactor In the of the S domain, particularly the and greatly the These diverse four the of the substrate-binding 1 and and in the highly of the substrate-binding for the from a to the cholesterol and oligosaccharides. The of ZmCKX, BsCOX2, PpPCMH, and PsVAO, the of the substrate-binding and in a E. A. A. J. Biol. 2004; PubMed Scopus Google Scholar, A. J. Biol. 2003; PubMed Scopus Google Scholar, L.M. Chen PubMed Scopus Google Scholar, van van A. J. Biol. PubMed Scopus Google Scholar). In GOOX has an open carbohydrate-binding to oligosaccharides. and in Sugar to the structural have been into D. Protein Sci. PubMed Scopus Google Scholar). Interestingly, does not with enzyme For example, GOOX and belong to the GOX, POX, CDH, and belong to the of the at the of the isoalloxazine ring is conserved the that of the is on the these structures of from the glucose oxidation is flavin substrate-binding In addition, of the have a toward covalent flavinylation at the 8α-methyl group for of flavinylation M. N.S. Protein Sci. 1998; PubMed Scopus Google Scholar), of the prefer a This may that the FAD-binding flavinylation to the for of its Interestingly, from a similar structural sugar oxidases residues for with the substrates. the GOX, POX, and catalyze the oxidation of glucose, and a similar F domain; their S domains lack conserved carbohydrate-interacting residues except for the conserved histidine as a general base G. G. A. C. J. Biol. 2003; PubMed Scopus Google Scholar, G. S. J. Schomburg D. Acta Crystallogr. Sect. D. 1999; 55: PubMed Scopus Google Scholar, C. D. C. J. Biol. 2004; PubMed Scopus Google Scholar). the although GOOX NlNEC5, CcHEOX, and HaCHOX in substrate specificity and F domain, the protein sequences of their S domains are quite diverse The and Tyr429 in GOOX, at the may conserved in NlNEC5, CcHEOX, and However, the other residues including and apparently are not these sugar oxidases have a similar oxidation substrate in to the mono- and disaccharides, with from 50 to 30 We F. Fan and J. W. for in structural using