Novel carbohydrate-binding module of beta-1,3-xylanase from a marine bacterium, Alcaligenes sp. strain XY-234

A beta-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp. strain XY-234, has been cloned and sequenced. txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the beta-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed beta-1,3-xylan but not other polysaccharides such as beta-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or beta-1,4-mannan. TxyA was capable of binding specifically to beta-1,3-xylan. The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the K(d) and the maximum amount of protein bound to beta-1,3-xylan were 4.2 microM and 18.2 micromol/g of beta-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of beta-1,3-xylan.     

An exo-beta-1,3-galactanase having a novel beta-1,3-galactan-binding module from Phanerochaete chrysosporium

An exo-beta-1,3-galactanase gene from Phanerochaete chrysosporium has been cloned, sequenced, and expressed in Pichia pastoris. The complete amino acid sequence of the exo-beta-1,3-galactanase indicated that the enzyme consists of an N-terminal catalytic module with similarity to glycoside hydrolase family 43 and an additional unknown functional domain similar to carbohydrate-binding module family 6 (CBM6) in the C-terminal region. The molecular mass of the recombinant enzyme was estimated as 55 kDa based on SDS-PAGE. The enzyme showed reactivity only toward beta-1,3-linked galactosyl oligosaccharides and polysaccharide as substrates but did not hydrolyze beta-1,4-linked galacto-oligosaccharides, beta-1,6-linked galacto-oligosaccharides, pectic galactan, larch arabinogalactan, arabinan, gum arabic, debranched arabinan, laminarin, soluble birchwood xylan, or soluble oat spelled xylan. The enzyme also did not hydrolyze beta-1,3-galactosyl galactosaminide, beta-1,3-galactosyl glucosaminide, or beta-1,3-galactosyl arabinofuranoside, suggesting that it specifically cleaves the internal beta-1,3-linkage of two galactosyl residues. High performance liquid chromatographic analysis of the hydrolysis products showed that the enzyme produced galactose from beta-1,3-galactan in an exo-acting manner. However, no activity toward p-nitrophenyl beta-galactopyranoside was detected. When incubated with arabinogalactan proteins, the enzyme produced oligosaccharides together with galactose, suggesting that it is able to bypass beta-1,6-linked galactosyl side chains. The C-terminal CBM6 did not show any affinity for known substrates of CBM6 such as xylan, cellulose, and beta-1,3-glucan, although it bound beta-1,3-galactan when analyzed by affinity electrophoresis. Frontal affinity chromatography for the CBM6 moiety using several kinds of terminal galactose-containing oligosaccharides as the analytes clearly indicated that the CBM6 specifically interacted with oligosaccharides containing a beta-1,3-galactobiose moiety. When the degree of polymerization of galactose oligomers was increased, the binding affinity of the CBM6 showed no marked change.     

Purification and characterization of beta-1,3-xylanase from a marine bacterium, Vibrio sp. XY-214

beta-1,3-Xylanase was purified to gel electrophoretic homogeneity and 83-fold from a cell-free culture fluid of Vibrio sp. XY-214 by ammonium sulfate precipitation and successive chromatographies. The enzyme had a pl of 3.6 and a molecular mass of 52 kDa. The enzyme had the highest level of activity at pH 7.0 and 37 degrees C. The enzyme activity was completely inhibited by Cu2+, Hg2+, and N-bromosuccinimide. The enzyme hydrolyzed beta-1,3-xylan to produce mainly xylotriose and xylobiose but did not act on xylobiose, p-nitrophenyl-beta-D-xyloside, beta-1,4-xylan, beta-1,3-glucan, or carboxymethyl cellulose.     

Cloning, sequencing, and expression in Escherichia coli of the new gene encoding beta-1,3-xylanase from a marine bacterium, Vibrio sp. strain XY-214

The Vibrio sp. strain XY-214 beta-1,3-xylanase gene cloned in Escherichia coli DH5alpha consisted of an open reading frame of 1, 383 nucleotides encoding a protein of 460 amino acids with a molecular mass of 51,323 Da and had a signal peptide of 22 amino acids. The transformant enzyme hydrolyzed beta-1,3-xylan to produce several xylooligosaccharides.     

The first crystal structure of a family 31 carbohydrate-binding module with affinity to beta-1,3-xylan

Here, we present the crystal structure of the family 31 carbohydrate-binding module (CBM) of beta-1,3-xylanase from Alcaligenes sp. strain XY-234 (AlcCBM31) determined at a resolution of 1.25A. The AlcCBM31 shows affinity with only beta-1,3-xylan. The AlcCBM31 molecule makes a beta-sandwich structure composed of eight beta-strands with a typical immunoglobulin fold and contains two intra-molecular disulfide bonds. The folding topology of AlcCBM31 differs from that of the large majority of other CBMs, in which eight beta-strands comprise a beta-sandwich structure with a typical jelly-roll fold. AlcCBM31 shows structural similarity with CBM structures of family 34 and family 9, which also adopt structures based on immunoglobulin folds.     

Purification and characterization of beta-1,3-xylanase from a marine bacterium,Alcaligenes sp. XY-234

A beta-1,3-xylanase-producing bacterium, Alcaligenes sp. XY-234, was isolated from the marine environment. The organism produced endo-1,3-beta-xylanase at a high level in the culture fluid. The enzyme was purified 292-fold by ammonium sulfate precipitation and several column chromatographies. The final enzyme preparation appeared to be homogeneous on disc gel electrophoresis and SDS-PAGE with a molecular mass of 59 kDa, and the pI was 4.0. The enzyme hydrolyzed beta-1,3-xylan and larger xylooligosaccharides than xylobiose to give several xylooligosaccharides, but it could not hydrolyze xylobiose, p-nitrophenyl-beta-D-xyloside, and beta-1,4-xylan. The Km of the enzyme was 4.0 mg/ml. Optimal pH and temperature were 7.5 and 40 degrees C, respectively. It was stable from pH 6.0 to 10 and at a temperature of less than 40 degrees C. The enzyme was strongly inhibited by 1 mM HgCl(2)., AlCl(3), CuCl(2), FeCl(3), HgCl(2), Pb(CH(3)COO) (2), and N-bromosuccinimide.     

Characterization of an exo-beta-1,3-galactanase from Clostridium thermocellum

A gene encoding an exo-beta-1,3-galactanase from Clostridium thermocellum, Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-beta-1,3-galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50 degrees C and catalyzes hydrolysis only of beta-1,3-linked galactosyl oligosaccharides and polysaccharides. High-performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from beta-1,3-galactan in an exo-acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a beta-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-beta-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including beta-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a beta-1,3-galactobiose, beta-1,4-galactosyl glucose, or beta-1,4-galactosyl N-acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward beta-1,3-galactan and alpha-l-arabinofuranosidase-treated AGP.     

Crystal structure of truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase in complex with beta-1,3-1,4-cellotriose

Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fsbeta-glucanase) catalyzes the specific hydrolysis of beta-1,4 glycosidic bonds adjacent to beta-1,3 linkages in beta-D-glucans or lichenan. This is the first report to elucidate the crystal structure of a truncated Fsbeta-glucanase (TFsbeta-glucanase) in complex with beta-1,3-1,4-cellotriose, a major product of the enzyme reaction. The crystal structures, at a resolution of 2.3 angstroms, reveal that the overall fold of TFsbeta-glucanase remains virtually unchanged upon sugar binding. The enzyme accommodates five glucose residues, forming a concave active cleft. The beta-1,3-1,4-cellotriose with subsites -3 to -1 bound to the active cleft of TFsbeta-glucanase with its reducing end subsite -1 close to the key catalytic residues Glu56 and Glu60. All three subsites of the beta-1,3-1,4-cellotriose adopted a relaxed C(1)4 conformation, with a beta-1,3 glycosidic linkage between subsites -2 and -1, and a beta-1,4 glycosidic linkage between subsites -3 and -2. On the basis of the enzyme-product complex structure observed in this study, a catalytic mechanism and substrate binding conformation of the active site of TFsbeta-glucanase is proposed.     

Purification and Properties of beta-1, 4-Xylanase from Aeromonas caviae W-61

Aeromonas caviae W-61, which was isolated from water samples at the Faculty of Agriculture, Tohoku University, produced beta-1, 4-xylanase (1,4-beta-d-xylan xylanohydrolase; EC 3.2.1.8) extracellularly. The xylanase was purified to homogeneity by using DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-100 column chromatographies. The molecular weight of the purified enzyme was estimated to be 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was 9.2. The optimal pH and temperature for the activity of the enzyme were 7.0 and 55 degrees C, respectively. The enzyme was stable at pH 7.0 at temperatures of up to 50 degrees C. As enzymatic products, various xylo-oligosaccharides such as xylobiose, xylotriose, xylotetraose, and xylopentaose were formed, and only a small amount of xylose was detected. The purified enzyme did not hydrolyze starch, cellulose, carboxymethylcellulose, or beta-1, 3-xylan.     

Enzymatic synthesis of 4-methylumbelliferyl (1-->3)-beta-D-glucooligosaccharides-new substrates for beta-1,3-1,4-D-glucanase

The transglycosylation reactions catalyzed by beta-1,3-D-glucanases (laminaranases) were used to synthesize a number of 4-methylumbelliferyl (MeUmb) (1-->3)-beta-D-gluco-oligosaccharides having the common structure [beta-D-Glcp-(1-->3)](n)-beta-D-Glcp-MeUmb, where n=1-5. The beta-1,3-D-glucanases used were purified from the culture liquid of Oerskovia sp. and from a homogenate of the marine mollusc Spisula sachalinensis. Laminaran and curdlan were used as (1-->3)-beta-D-glucan donor substrates, while MeUmb-beta-D-glucoside (MeUmbGlcp) was employed as a transglycosylation acceptor. Modification of [beta-D-Glcp-(1-->3)](2)-beta-D-Glcp-MeUmb (MeUmbG(3)) gives 4,6-O-benzylidene-D-glucopyranosyl or 4,6-O-ethylidene-D-glucopyranosyl groups at the non-reducing end of artificial oligosaccharides. The structures of all oligosaccharides obtained were solved by 1H and 13C NMR spectroscopy and electrospray tandem mass spectrometry. The synthetic oligosaccharides were shown to be substrates for a beta-1,3-1,4-D-glucanase from Rhodothermus marinus, which releases MeUmb from beta-di- and beta-triglucosides and from acetal-protected beta-triglucosides. When acting upon substrates with d.p.>3, the enzyme exhibits an endolytic activity, primarily cleaving off MeUmbGlcp and MeUmbG(2).     

Occurrence of poly-N-acetyllactosamine synthesis in Sf-9 cells upon transfection of individual human beta-1,4-galactosyltransferase I, II, III, IV, V and VI cDNAs

Lectin blot analysis of membrane glycoprotein samples from Sf-9 cells upon transfection of individual human beta-1,4-galactosyltransferase (beta-1,4-GalT) I, II, III, IV, V et VI cDNAs showed that the endogenous N-linked oligosaccharides are galactosylated (Guo et al., Glycobiology (2001), in press). Further analysis revealed that membrane glycoprotein samples from all the gene-transfected cells are also reactive to Lycopersicon esculentum agglutinin (LEA) et Datura stramonium agglutinin (DSA), both of which bind to oligosaccharides with poly-N-acetyllactosamine chains while no lectin reactive protein bands are detected when blots are pretreated with a mixture of diplococcal beta-1,4-galactosidase et jack bean beta-N-acetylhexosaminidase or N-glycanase. Analysis of endo-beta-galactosidase-digestion products revealed the presence of the Gal1-->GlcNAc1-->Gal and/or GlcNAc1-->Gal structures in the gene-transfected cells. When the homogenates of the gene-transfected cells were used as enzyme sources towards oligosaccharides with the GlcNAc beta 1-->(3Gal beta 1-->4GlcNAc)(1-3) structures, human recombinant beta-1,4-GalTs I et II galactosylated these oligosaccharides more effectively than other beta-1,4-GalTs. These results indicate that beta-1,4-GalTs I-VI can synthesize poly-N-acetyllactosamine chains with beta-1,3-N-acetylglucosaminyltransferase.     

The family 11 carbohydrate-binding module of Clostridium thermocellum Lic26A-Cel5E accommodates beta-1,4- and beta-1,3-1,4-mixed linked glucans at a single binding site

Modular glycoside hydrolases that attack recalcitrant polymers generally contain noncatalytic carbohydrate-binding modules (CBMs), which play a critical role in the action of these enzymes by localizing the appended catalytic domains onto the surface of insoluble polysaccharide substrates. Type B CBMs, which recognize single polysaccharide chains, display ligand specificities that are consistent with the substrates hydrolyzed by the associated catalytic domains. In enzymes that contain multiple catalytic domains with distinct substrate specificities, it is unclear how these different activities influence the evolution of the ligand recognition profile of the appended CBM. To address this issue, we have characterized the properties of a family 11 CBM (CtCBM11) in Clostridium thermocellum Lic26A-Cel5E, an enzyme that contains GH5 and GH26 catalytic domains that display beta-1,4- and beta-1,3-1,4-mixed linked endoglucanase activity, respectively. Here we show that CtCBM11 binds to both beta-1,4- and beta-1,3-1,4-mixed linked glucans, displaying K(a) values of 1.9 x 10(5), 4.4 x 10(4), and 2 x 10(3) m(-1) for Glc-beta1,4-Glc-beta1,4-Glc-beta1,3-Glc, Glc-beta1,4-Glc-beta1,4-Glc-beta1,4-Glc, and Glc-beta1,3-Glc-beta1,4-Glc-beta1,3-Glc, respectively, demonstrating that CBMs can display a preference for mixed linked glucans. To determine whether these ligands are accommodated in the same or diverse sites in CtCBM11, the crystal structure of the protein was solved to a resolution of 1.98 A. The protein displays a beta-sandwich with a concave side that forms a potential binding cleft. Site-directed mutagenesis revealed that Tyr(22), Tyr(53), and Tyr(129), located in the putative binding cleft, play a central role in the recognition of all the ligands recognized by the protein. We propose, therefore, that CtCBM11 contains a single ligand-binding site that displays affinity for both beta-1,4- and beta-1,3-1,4-mixed linked glucans.     

Isolation and overexpression of a gene encoding an extracellular beta-(1,3-1,4)-glucanase from Streptococcus bovis JB1.

Streptococcus bovis JB1 was found to produce a 25-kDa extracellular enzyme active against beta-(1,3-1,4)-glucans. A gene was isolated encoding a specific beta-(1,3-1,4)-glucanase that corresponds to this size and belongs to glycoside hydrolase family 16. A 4- to 10-fold increase in supernatant beta-glucanase activity was obtained when the cloned beta-glucanase gene was reintroduced into S. bovis JB1 by use of constructs based on the plasmid vector pTRW10 or pIL253. The beta-(1,3-1,4)-glucanase gene was also expressed upon introduction of the pTRW10 construct pTRWL1R into Lactococcus lactis IL2661 and Enterococcus faecalis JH2-SS, although extracellular activity was 8- to 50-fold lower than that in S. bovis JB1. The beta-(1,3-1,4)-glucanase purified from the culture supernatant of S. bovis JB1 carrying pTRWL1R showed a K(m) of 2.8 mg per ml and a Vmax of 338 mumol of glucose equivalents per min per mg of protein with barley beta-glucan as the substrate. The S. bovis beta-(1,3-1,4)-glucanase may contribute to the ability of this bacterium to utilize starch by degrading structural polysaccharides present in endosperm cell walls.     

Properties of a thermoactive beta-1,3-1,4-glucanase (lichenase) from Clostridium thermocellum expressed in Escherichia coli

A Clostridium thermocellum gene (licB) encoding a thermoactive 1,3-1,4-beta-glucanase (lichenase) with a molecular weight of about 35,000 was localized on a 1.5-kb DNA fragment by cloning and expression in E. coli. The enzyme acts on beta-glucans with alternating beta-1,3- and beta-1,4-linkages such as barley beta-glucan and lichenan, but not on beta-glucans containing only 1,3- or 1,4-glucosidic bonds. It is active over a broad pH range (pH 5-12) and has a temperature optimum around 80 degrees C. The C. thermocellum lichenase is unusually resistant against inactivation by heat, ethanol or ionic detergents. These properties make the enzyme highly suitable for industrial application in the mashing process of beer brewing.     

Isolation of extracellular 28- and 42-kilodalton beta-1,3-glucanases and comparison of three beta-1,3-glucanases produced by Bacillus circulans IAM1165.

Bacillus circulans IAM1165 produces three major extracellular beta-1,3-glucanases (molecular masses, 28, 42, and 91 kDa) during the stationary phase of growth. The 28- and 42-kDa enzymes were purified to homogeneity from the culture supernatant in this study. The properties of these two enzymes were examined, together with those of the 91-kDa enzyme previously isolated. The enzymatic properties of the 28- and 42-kDa beta-1,3-glucanases closely resemble each other. The enzymes belong to a category of endo type 1,3-beta-D-glucan glucanohydrolases. The enzymes were active at pH 4.0 to 7.0. The optimum temperature of the reactions was 60 degrees C when laminarin (a soluble beta-1,3-glucan) was used as the substrate at pH 7.0. The enzymes hydrolyzed barley glucan and lichenan (beta-1,3-1,4-glucans) more effectively than laminarin. Of the three enzymes, the 42-kDa enzyme lysed fungal cell walls the most effectively.     

Development of beta-1,3-glucanase activity in germinated tomato seeds

Laminarin-hydrolysing activity developed in the endosperm of tomato (Lycopersicon esculentum) seeds following germination. The enzyme was basic (pI>10) and the apparent molecular mass was estimated to be 35 kDa by SDS-PAGE. It was specific for linear beta-1,3-glucan substrates. Laminarin was hydrolysed by the enzyme to yield a mixture of oligoglucosides, indicating that the enzyme had an endo-action pattern. Thus, the enzyme was identified as beta-1,3- endoglucanase (EC 3.2.1.39). The activity of the enzyme developed in the endosperm after radicle protrusion (germination) had occurred and the enzyme activity was localized exclusively in the micropylar region of the endosperm where the radicle had penetrated. When the lateral endosperm region, where no induction of the enzyme occurred, was wounded (cut or punctured), there was a marked enhancement of beta-1,3-glucanase activity. Thus the post-germinative beta-1, 3-glucanase activity in the micropylar endosperm portion might be brought about by wounding resulting from endosperm rupture by radicle penetration.     

A simple enzymatic synthesis of 4-nitrophenyl beta-1,4-D-xylobioside, a chromogenic substrate for assay and differentiation of endoxylanases

A simple procedure has been elaborated for preparation of 4-nitrophenyl beta-d-xylopyranosyl-1,4-beta-d-xylopyranoside (NPX(2)), a chromogenic substrate of some endo-beta-1,4-xylanases. The procedure is based on a self-transfer reaction from 4-nitrophenyl beta-d-xylopyranoside catalyzed by an Aureobasidium pullulans and Aspergillus niger beta-xylosidases. Both enzymes catalyzed only the formation of 4-nitrophenyl glycosides of beta-1,4-xylobiose with a small admixture of 4-nitrophenyl glycoside of beta-1,3-xylobiose. The highest yields of the NPX(2) (19.4%) was obtained at pH 5.5. The removal of the beta-1,3-isomer from NPX(2) is not necessary for quantification of endo-beta-1,4-xylanase activity since it is not attacked by endo-beta-1,4-xylanases. In contrast to GH family 5 xylanase from Erwinia chrysanthemi, which did not attack NPX(2), all family 10 and 11 xylanases cleaved the chromogenic substrate exclusively between xylobiose and the aromatic aglycone. Significant differences in the K(m) values of GH10 and GH11 xylanases suggested that activities of these enzymes could be selectively quantified in the mixtures using various concentrations of NPX(2). Moreover, NPX(2) could serve as an ideal substrate to follow the interaction of endo-beta-1,4-xylanases with various xylanase inhibitors.     

Structural studies of the mix-linked beta-(1-->3)/beta-(1-->4)-D-xylans from the cell wall of Palmaria palmata (Rhodophyta)

The structure and organization of Palmaria palmata cell walls, which are largely involved in biological and physiological functions as well as in biotechnological and food applications of this red marine alga, are principally assumed by the interactions and linkages of major mix-linked beta-(1-->3)/beta-(1-->4)-D-xylans. These partly acidic polysaccharides are essentially held in the cell wall by H-bonds. The location of the acid groups and the distribution of 1-->3-linkage were studied following the endo-beta-(1,4)-xylanase hydrolysis of sequentially extracted xylans, and fine analysis of the oligosaccharides produced by anion exchange chromatography, high performance anion exchange chromatography (HPAEC)-PAD, nuclear magnetic resonance (NMR) and electrospray ion trap mass spectrometry (ESI-MS) techniques. The results indicate that the acidity of the xylans was related to potential linkages to sulfated and/or phosphorylated xylogalactoprotein complexes. H-bonding of the mix-linked xylans involved a regular 1,3-linkages distribution idealized in a pentameric repeating structure (one 1,3-linkage and four 1,4-linkages). Furthermore, MS analysis of the xylo-oligosaccharides revealed a substitution of the mix-linked xylans by a non-osidic component of 175 g mol(-1). The presence of this substituent and of the proposed covalent linkage between the mix-linked xylans and charged glycoproteins are discussed with regard to the polysaccharides interactions in P. palmata cell walls.     

Hydrolase and transferase activities of the beta-1,3-exoglucanase of Candida albicans.

The exo-beta-1,3-glucanase of Candida albicans (Exg) has a marked specificity for beta-1,3-glucosidic linkages as judged by the kinetic constants for p-nitophenyl beta-glucoside, beta-linked disaccharides of glucose (laminaribiose, gentiobiose, and cellobiose), oligosaccharides of the laminari series, laminarin and pustulan. The kcat/Km ratios for a series of laminari oligosaccharides from -biose to -heptaose showed that Exg has an extended substrate-binding site which contains at least five binding sites for sugar residues. Binding at position +2 (the third sugar residue) increases the kcat twofold while positions +3 and +4 lower the Km value further and thereby increase the catalytic efficiency. Exg catalyses an efficient transglucosylation reaction with high concentrations of laminari-oligosaccharides which specifically form beta-1,3 linkages and with yields up to 50%. The rate of the transglucosylation is concentration-dependent and can be more than 10 times faster than the hydrolytic reaction with excess donor substrates such as laminaritriose and laminarihexaose. The kinetics of Exg and the predicted substrate-binding site for up to five sugar residues are consistent with a recent structural analysis of the enzyme-binding site.