Structure-based engineering of glucose specificity in a family 10 xylanase from Streptomyces olivaceoviridis E-86

Substrate specificity is one of the most important functional property of enzymes. We use family 10 xylanase from Streptomyces olivaceoviridis as a model for substrate specificity of glycoside hydrolases. Seven variants were initially designed to change the preference from xylose to glucose at substrate binding subsites ?2 and ?1. The known mobility of Trp at the ?1 subsite and the influence of its environment, which is different in subset 1 and subset 2 family 10 enzymes, were taken into account in variant design. Q88A/R275A had the best ratio of p-nitrophenyl cellobioside vs p-nitrophenyl xylobioside hydrolyzing activity in the first series of variants. The crystal structure shows a movement of Trp274 compared to the native, as a result of loss of interaction with the long side chain of Arg275. The movement creates extra space for the hydroxymethyl of glucose, resulting in improved K_m on glucose derived substrates, while the negative effect on k_cat is compensated by the Q88A mutation, which also contributes to a further reduction of K_m. Further mutagenesis based on the Q88A/R275A variant resulted in 5.2 times improvement compared to the wild-type p-nitrophenyl cellobioside hydrolyzing activity, which is the best improvement obtained so far for an engineered xylanase.     

Crystal structure of Streptomyces olivaceoviridis E-86 beta-xylanase containing xylan-binding domain

Xylanases hydrolyse the beta-1,4-glycosidic bonds within the xylan backbone and belong to either family 10 or 11 of the glycoside hydrolases, on the basis of the amino acid sequence similarities of their catalytic domains. Generally, xylanases have a core catalytic domain, an N and/or C-terminal substrate-binding domain and a linker region. Until now, X-ray structural analyses of family 10 xylanases have been reported only for their catalytic domains and do not contain substrate-binding domains. We have determined the crystal structure of a family 10 xylanase containing the xylan-binding domain (XBD) from Streptomyces olivaceoviridis E-86 at 1.9 A resolution. The catalytic domain comprises a (beta/alpha)(8)-barrel topologically identical to other family 10 xylanases. XBD has three similar subdomains, as suggested from a triple-repeat sequence, which are assembled against one another around a pseudo-3-fold axis, forming a galactose-binding lectin fold similar to ricin B-chain. The Gly/Pro-rich linker region connecting the catalytic domain and XBD is not visible in the electron density map, probably because of its flexibility. The interface of the two domains in the crystal is hydrophilic, where five direct hydrogen bonds and water-mediated hydrogen bonds exist. The sugar-binding residues seen in ricin/lactose complex are spatially conserved among the three subdomains in XBD, suggesting that all of the subdomains in XBD have the capacity to bind sugars. The flexible linker region enables the two domains to move independently and may provide a triple chance of substrate capturing and catalysis. The structure reported here represents an example where the metabolic enzyme uses a ricin-type lectin motif for capturing the insoluble substrate and promoting catalysis.     

Crystal structures of decorated xylooligosaccharides bound to a family 10 xylanase from Streptomyces olivaceoviridis E-86

The family 10 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A) consists of a GH10 catalytic domain, which is joined by a Gly/Pro-rich linker to a family 13 carbohydrate-binding module (CBM13) that interacts with xylan. To understand how GH10 xylanases and CBM13 recognize decorated xylans, the crystal structure of SoXyn10A was determined in complex with alpha-l-arabinofuranosyl- and 4-O-methyl-alpha-d-glucuronosyl-xylooligosaccharides. The bound sugars were observed in the subsites of the catalytic cleft and also in subdomains alpha and gamma of CBM13. The data reveal that the binding mode of the oligosaccharides in the active site of the catalytic domain is entirely consistent with the substrate specificity and, in conjunction with the accompanying paper, demonstrate that the accommodation of the side chains in decorated xylans is conserved in GH10 xylanases of SoXyn10A against arabinoglucuronoxylan. CBM13 was shown to bind xylose or xylooligosaccharides reversibly by using nonsymmetric sugars as the ligands. The independent multiple sites in CBM13 may increase the probability of substrate binding.     

Purification and characterization of a family G/11 beta-xylanase from Streptomyces olivaceoviridis E-86

A beta-xylanase (GXYN) was purified from the culture filtrate of Streptomyces olivaceoviridis E-86 by successive chromatography on DE-52, CM-Sepharose and Superose 12. The molecular mass of the xylanase was estimated to be 23 kDa, indicating that the enzyme consists of a catalytic domain only. The enzyme displayed an optimum pH of 6, a temperature optimum of 60 degrees C, a pH stability range from 2 to 11 and thermal stability up to 40 degrees C. The N-terminal amino acid sequence of GXYN was A-T-V-I-T-T-N-Q-T-G-T-N-N-G-I-Y-Y-S-F-W-, and sharing a high degree of similarity with the N-terminal sequence of xylanases B and C from Streptomyces lividans, indicating GXYN belongs to family G/11 of glycoside hydrolases. GXYN was inferior to xylanase B from Streptomyces lividans in the hydrolysis of insoluble xylan because of its lack of a xylan binding domain.     

Structure and function of a family 10 beta-xylanase chimera of Streptomyces olivaceoviridis E-86 FXYN and Cellulomonas fimi Cex

The catalytic domain of xylanases belonging to glycoside hydrolase family 10 (GH10) can be divided into 22 modules (M1 to M22; Sato, Y., Niimura, Y., Yura, K., and Go, M. (1999) Gene (Amst.) 238, 93-101). Inspection of the crystal structure of a GH10 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A) revealed that the catalytic domain of GH10 xylanases can be dissected into two parts, an N-terminal larger region and C-terminal smaller region, by the substrate binding cleft, corresponding to the module border between M14 and M15. It has been suggested that the topology of the substrate binding clefts of GH10 xylanases are not conserved (Charnock, S. J., Spurway, T. D., Xie, H., Beylot, M. H., Virden, R., Warren, R. A. J., Hazlewood, G. P., and Gilbert, H. J. (1998) J. Biol. Chem. 273, 32187-32199). To facilitate a greater understanding of the structure-function relationship of the substrate binding cleft of GH10 xylanases, a chimeric xylanase between SoXyn10A and Xyn10A from Cellulomonas fimi (CfXyn10A) was constructed, and the topology of the hybrid substrate binding cleft established. At the three-dimensional level, SoXyn10A and CfXyn10A appear to possess 5 subsites, with the amino acid residues comprising subsites -3 to +1 being well conserved, although the +2 subsites are quite different. Biochemical analyses of the chimeric enzyme along with SoXyn10A and CfXyn10A indicated that differences in the structure of subsite +2 influence bond cleavage frequencies and the catalytic efficiency of xylooligosaccharide hydrolysis. The hybrid enzyme constructed in this study displays fascinating biochemistry, with an interesting combination of properties from the parent enzymes, resulting in a low production of xylose.     

An investigation of the nature and function of module 10 in a family F/10 xylanase FXYN of Streptomyces olivaceoviridis E-86 by module shuffling with the Cex of Cellulomonas fimi and by site-directed mutagenesis

Although the amino acid homology in the catalytic domain of FXYN xylanase from Streptomyces olivaceoviridis E-86 and Cex xylanase from Cellulomonas fimi is only 50%, an active chimeric enzyme was obtained by replacing module 10 in FXYN with module 10 from Cex. In the family F/10 xylanases, module 10 is an important region as it includes an acid/base catalyst and a substrate binding residue. In FXYN, module 10 consists of 15 amino acid residues, while in Cex it consists of 14 amino acid residues. The Km and kcat values of the chimeric xylanase FCF-C10 for PNP-xylobioside (PNP-X2) were 10-fold less than those for FXYN. CD spectral data indicated that the structure of the chimeric enzyme was similar to that of FXYN. Based on the comparison of the amino acid sequences of FXYN and Cex in module 10, we constructed four mutants of FXYN. When D133 or S135 of FXYN was deleted, the kinetic properties were not changed from those of FXYN. By deletion of both D133 and S135, the Km value for PNP-X2 decreased from the 2.0 mM of FXYN to 0.6 mM and the kcat value decreased from the 20 s(-1) of FXYN to 8.7 s(-1). Insertion of Q140 into the doubly deleted mutant further reduced the Km value to 0.3 mM and the kcat value to 3.8 s(-1). These values are close to those for the chimeric enzyme FCF-C10. These results indicate that module 10 itself is able to accommodate changes in the sequence position of amino acids which are critical for enzyme function. Since changes of the spatial position of these amino acids would be expected to result in enzyme inactivation, module 10 must have some flexibility in its tertiary structure. The structure of module 10 itself also affects the substrate specificity of the enzyme.     

Module shuffling of a family F/10 xylanase: replacement of modules M4 and M5 of the FXYN of Streptomyces olivaceoviridis E-86 with those of the Cex of Cellulomonas fimi

To facilitate an understanding of structure-function relationships, chimeric xylanases were constructed by module shuffling between the catalytic domains of the FXYN from Streptomyces olivaceoviridis E-86 and the Cex from Cellulomonas fimi. In the family F/10 xylanases, the modules M4 and M5 relate to substrate binding so that modules M4 and M5 of the FXYN were replaced with those of the Cex and the chimeric enzymes denoted FCF-C4, FCF-C5 and FCF-C4,5 were constructed. The k(cat) value of FCF-C5 for p-nitrophenyl-beta-D-cellobioside was similar to that of the FXYN (2.2 s(-1)); however, the k(cat) value of FCF-C4 for p-nitrophenyl-beta-D-cellobioside was significantly higher (7.0 s(-1)). The loss of the hydrogen bond between E46 and S22 or the presence of the I49W mutation would be expected to change the position of Q88, which plays a pivotal role in discriminating between glucose and xylose, resulting in the increased k(cat) value observed for FCF-C4 acting on p-nitrophenyl-beta-D-cellobioside since module M4 directly interacts with Q88. To investigate the synergistic effects of the different modules, module M10 of the FCF-C4 chimera was replaced with that of the Cex. The effects of replacement of module M4 and M10 were almost additive with regard to the K:(m) and k(cat) values.     

Subunit composition of a large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86

A xylanolytic complex (xylanosome) was isolated from Streptomyces olivaceoviridis E-86 grown on corncob xylan. The isolated xylanosome exhibited a high molecular mass of approximately 3.8 x 10(7) Da (weight average) using size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS), and was composed of at least 8 subunits with a mass range from 12 to 60 kDa. When a SDS-polyacrylamide gel zymogram was examined, the subunits of 47, 35, 32, and 23 kDa were found to have xylanase activity, while the 30-kDa subunit had CMCase activity. According to N-terminal sequence analyses, the 47- and 23-kDa subunits were found to be identical to the two reported xylanases, namely FXYN and GXYN, of S. olivaceoviridis E-86. Both the 35- and 32-kDa subunits were found to be truncated forms of the intact FXYN xylanase that possibly resulted from the degradation by proteases. The 15-kDa subunit consisted solely the xylan-binding domain of the FXYN xylanase. The purified xylanosome appeared to bind partially to xylan and poorly to Avicel.     

Substrate specificity in glycoside hydrolase family 10. Structural and kinetic analysis of the Streptomyces lividans xylanase 10A

Endoxylanases are a group of enzymes that hydrolyze the beta-1, 4-linked xylose backbone of xylans. They are predominantly found in two discrete sequence families known as glycoside hydrolase families 10 and 11. The Streptomyces lividans xylanase Xyl10A is a family 10 enzyme, the native structure of which has previously been determined by x-ray crystallography at a 2.6 A resolution (Derewenda, U., Swenson, L., Green, R., Wei, Y., Morosoli, R., Shareck, F., Kluepfel, D., and Derewenda, Z. S. (1994) J. Biol. Chem. 269, 20811-20814). Here, we report the native structure of Xyl10A refined at a resolution of 1.2 A, which reveals many features such as the rare occurrence of a discretely disordered disulfide bond between residues Cys-168 and Cys-201. In order to investigate substrate binding and specificity in glycoside hydrolase family 10, the covalent xylobiosyl enzyme and the covalent cellobiosyl enzyme intermediates of Xyl10A were trapped through the use of appropriate 2-fluoroglycosides. The alpha-linked intermediate with the nucleophile, Glu-236, is in a (4)C(1) chair conformation as previously observed in the family 10 enzyme Cex from Cellulomonas fimi (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998) Biochemistry 37, 4751-4758). The different interactions of Xyl10A with the xylobiosyl and cellobiosyl moieties, notably conformational changes in the -2 and -1 subsites, together with the observed kinetics on a range of aryl glycosides, shed new light on substrate specificity in glycoside hydrolase family 10.     

Significant enhancement in the binding of p-nitrophenyl-β- -xylobioside by the E128H mutant F/10 xylanase from Streptomyces olivaceoviridis E-86

Mutagenesis studies were carried out to examine the effects of replacement of either the nucleophile Glu-236 or the acid/base Glu-128 residue of the F/10 xylanase by a His residue. To our surprise, the affinity for the p-nitrophenyl-β- -xylobioside substrate was increased by 10³-fold in the case of the mutant E128H enzyme compared with that of the wild-type F/10 xylanase. The catalytic activity of the mutant enzymes was low, despite the fact that the distance between the nucleophilic atom (an oxygen in the native xylanase and a nitrogen in the mutant) and the α-carbon was barely changed. Thus, the alteration of the acid/base functionality (Glu-128 to His mutation) provided a significantly favorable interaction within the E128H enzyme/substrate complex in the ground state, accompanying a reduction in the stabilization effect in the transition state.     

An enzyme-linked immunosorbent assay (ELISA) to determine the specificity of the sugar-binding protein NgcE, a component of the ABC transporter for N-acetylglucosamine in Streptomyces olivaceoviridis

The ATP-binding cassette (ABC) transporter Ngc for N-acetylglucosamine (GlcNAc) of the chitin-degrader Streptomyces olivaceoviridis comprises the solute-binding protein NgcE, which has highest affinity for GlcNAc and N,N'-diacetylchitobiose {(GlcNAc)2} and reduced affinity for longer chitooligomers. NgcE was used to develop a generally applicable enzyme-linked immunosorbent assay (ELISA) system. As a prerequisite, the reducing end of (GlcNAc)2 was coupled with the ethylamino group of 2-(4-aminophenyl)ethylamine. The resulting conjugate was linked with amino groups of bovine serum albumin (BSA) to gain the neoglycoprotein BSA-APEA-(GlcNAc)2, which was fixed to wells in microtitre-plates. The NgcE protein was shown to bind efficiently to the immobilized BSA-APEA-(GlcNAc)2. In competition assays, the affinity of NgcE was 1,000-fold higher for GlcNAc and (GlcNAc)2 than for (GlcNAc)3 and (GlcNAc)4. These results are consistent with those previously obtained by surface plasmon resonance. Since the ELISA method can be performed very rapidly at low cost, it should be an efficient general tool to determine the affinity of a ligand to its cognate solute-binding protein.     

Binding specificity and thermodynamics of a family 9 carbohydrate-binding module from Thermotoga maritima xylanase 10A

The C-terminal family 9 carbohydrate-binding module of xylanase 10A from Thermotoga maritima (CBM9-2) binds to amorphous cellulose, crystalline cellulose, and the insoluble fraction of oat spelt xylan. The association constants (K(a)) for adsorption to insoluble polysaccharides are 1 x 10(5) to 3 x 10(5) M(-1). Of the soluble polysaccharides tested, CBM9-2 binds to barley beta-glucan, xyloglucan, and xylan. CBM9-2 binds specifically to the reducing ends of cellulose and soluble polysaccharides, a property that is currently unique to this CBM. CBM9-2 also binds glucose, xylose, galactose, arabinose, cellooligosaccharides, xylooligosaccharides, maltose, and lactose, with affinities ranging from 10(3) M(-1) for monosaccharides to 10(6) M(-1) for disaccharides and oligosaccharides. Cellooligosaccharides longer than two glucose units do not bind with improved affinity, indicating that cellobiose is sufficient to occupy the entire binding site. In general, the binding reaction is dominated by favorable changes in enthalpy, which are partially compensated by unfavorable entropy changes.     

High-resolution crystal structures of the lectin-like xylan binding domain from Streptomyces lividans xylanase 10A with bound substrates reveal a novel mode of xylan binding

Carbohydrate-binding module (CBM) family 13 includes the "R-type" or "ricin superfamily" beta-trefoil lectins. The C-terminal CBM, CBM13, of xylanase 10A from Streptomyces lividans is a family 13 CBM that is not only structurally similar to the "R-type" lectins but also somewhat functionally similar. The primary function of CBM13 is to bind the polysaccharide xylan, but it retains the ability of the R-type lectins to bind small sugars such as lactose and galactose. The association of CBM13 with xylan appears to involve cooperative and additive participation of three binding pockets in each of the three trefoil domains of CBM13, suggesting a novel mechanism of CBM-xylan interaction. Thus, the interaction of CBM13 with sugars displays considerable plasticity for which we provide a structural rationale. The high-resolution crystal structure of CBM13 was determined by multiple anomalous dispersion from a complex of CBM13 with a brominated ligand. Crystal structures of CBM13 in complex with lactose and xylopentaose revealed two distinct mechanisms of ligand binding. CBM13 has retained its specificity for lactose via Ricin-like binding in all of the three classic trefoil binding pockets. However, CBM13 has the ability to bind either the nonreducing galactosyl moiety or the reducing glucosyl moiety of lactose. The mode of xylopentaose binding suggests adaptive mutations in the trefoil sugar binding scaffold to accommodate internal binding on helical polymers of xylose.     

The N-terminal family 22 carbohydrate-binding module of xylanase 10B of Clostridium themocellum is not a thermostabilizing domain

Xylanase Xyn10B from Clostridium thermocellum is a modular enzyme that contains two family 22 carbohydrate binding modules N- (CBM22-1) and C- (CBM22-2) terminal of the family 10 glycoside hydrolase catalytic domain (GH10). In a previous study, we showed that removal of CBM22-1 reduces the resistance to thermoinactivation of the enzyme suggesting that this module is a thermostabilizing domain. Here, we show that it is the module border on the N-terminal side of GH10 that confers resistance to thermoinactivation and to proteolysis. Therefore, CBM22-1 does not function as a thermostabilizing domain and the role for this apparently non-functional CBM remains elusive.     

Site-specific characterization of the association of xylooligosaccharides with the CBM13 lectin-like xylan binding domain from Streptomyces lividans xylanase 10A by NMR spectroscopy

Endo-beta-1,4-xylanase 10A (Xyn10A) from Streptomyces lividans includes an N-terminal catalytic module and a 130-residue C-terminal family 13 carbohydrate-binding module (CBM13). This latter domain adopts a beta-trefoil structure with three potential binding sites (alpha, beta, and gamma) for a variety of small sugars, xylooligosaccharides, and xylan polymers. To investigate the role of this multivalency in carbohydrate binding, we have used NMR spectroscopy to characterize the interaction of isolated CBM13 with a series of sugars. We have assigned resonances from the main chain nuclei of CBM13 using heteronuclear NMR experiments. Analysis of (15)N NMR relaxation data using the extended model free formalism reveals that CBM13 tumbles as an oblate ellipsoid (D( parallel)/D( perpendicular) = 0.80 +/- 0.02) and that its backbone is relatively rigid on the sub-nanosecond time scale. In particular, the three binding sites show no distinct patterns of increased internal mobility. Ligand-induced chemical shift changes in the (1)H-(15)N HSQC spectra of CBM13 were monitored as a function of increasing concentrations of L-arabinose, lactose, D-xylose, xylobiose, xylotetraose, and xylohexaose. Patterns of shift perturbations for well-resolved resonances demonstrate that all of these sugars associate independently with the three binding sites of CBM13. On the basis of the site-specific association constants derived from a quantitative analysis of these titration data, we show that L-arabinose, lactose, and D-xylose preferentially bind to the alpha site of CBM13, xylobiose binds equally well to all three sites, and xylotetraose and xylohexaose prefer binding to the beta site. Inspection of the crystallographic structure of CBM13 [Notenboom, V., Boraston, A. B., Williams, S. J., Kilburn, D. G., and Rose, D. R. (2002) Biochemistry 41, 4246-4254] provides a rationalization for these results.     

Improving the thermostability of a mesophilic family 10 xylanase,AuXyn10A,from Aspergillus usamii by in silico design

To improve the thermostability of a mesophilic GH family 10 xylanase, AuXyn10A, from Aspergillus usamii E001, its modification was performed by in silico design. Based on the comparison of B-factor values, a mutant xylanase ATXyn10 was predicted by substituting a segment YP from Tyr²⁵ to Pro³⁴ of AuXyn10A with the corresponding one from Asn²⁴ to Ala³² of TaXyn10, a thermophilic GH family 10 xylanase from Thermoascus aurantiacus. Analysis of a TaXyn10 crystal structure indicated that there is a close interaction between segments YP and FP. For that reason, another mutant xylanase ATXyn10^M was designed by mutating Ser²⁸⁶ and His²⁸⁸ of ATXyn10 into the corresponding Gly²⁸⁵ and Phe²⁸⁷ in the FP of TaXyn10. Then, two ATXyn10- and ATXyn10^M-encoding genes, ATxyn10 and ATxyn10 ^M, were expressed in Pichia pas toris GS115. The temperature optimum of recombinant (re) ATXyn10^M was 60 °C, 10 °C higher than that of reAuXyn10A. Its thermal inactivation half-life (t _1/2) at 55 °C was 10.4-fold longer than that of reAuXyn10A. As compared with reAuXyn10A, reATXyn10^M displayed a slight decrease in K _m value and a significant increase in V _max value from 6,267 to 8,870 U/mg.     

Structural basis for the substrate specificity of the feruloyl esterase domain of the cellulosomal xylanase Z from Clostridium thermocellum

Feruloyl esterases function in the cleavage of ferulic acid's bonds to arabinoxylan and pectin where the ferulic acid moieties cross-link the layers of polysaccharide chains within hemicellulose. This work presents the crystal structure of FAE_XynZ, the domain of Clostridium thermocellum's cellulosomal xylanase Z that displays feruloyl esterase activity. The structure was obtained via multiple isomorphous replacement with anomalous scattering (MIRAS) using three heavy atom derivatives and refined against X-ray diffraction data of up to 1.75 A resolution. The R-value of the final model was 0.187 (R(free) = 0.21). FAE_XynZ displays an eight-stranded alpha/beta-fold with the characteristic "catalytic triad" at the heart of the active site. To define the substrate specificity determinants of the enzyme, the crystal structures of FAE_XynZ and the inactive FAE_XynZ(S172A) mutant were determined in complexes with the feruloyl-arabinoxylans FAXX and FAX(3), respectively. In the complex crystals, the ferulic acid moieties are clearly recognizable and allowed identification of the hydrophobic binding pocket. The carbohydrate part of both substrates is not visible in either structure. The location of the putative carbohydrate binding-pocket was inferred based on the location and orientation of the adjacent ferulic acid molecule. Five of the six residues lining the pocket were found to be conserved in FAE A from Orpinomyces sp., which further supports the proposed role of these amino acids.