Aglycone Subsites Research Articles

Two-domain GH30 xylanase from human gut microbiota as a tool for enzymatic production of xylooligosaccharides: Crystallographic structure and a synergy with GH11 xylosidase

Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy. This includes valorization of hemicellulosic fraction of plant biomass, the second most abundant biopolymer from plant cell walls, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In this work, we conducted biochemical and biophysical characterization of a prokaryotic two-domain R. champanellensis xylanase from glycoside hydrolase (GH) family 30 (RcXyn30A), and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH10 and GH11 families and a GH11 xylobiohydrolase. RcXyn30A liberates mainly long monoglucuronylated xylooligosaccharides and is inefficient in cleaving unbranched oligosaccharides. Crystallographic structure of RcXyn30A catalytic domain was solved and refined to 1.37 Å resolution. Structural analysis of the catalytic domain releveled that its high affinity for glucuronic acid substituted xylan is due to the coordination of the substrate decoration by several hydrogen bonds and ionic interactions in the subsite −2. Furthermore, the protein has a larger β5-α5 loop as compared to other GH30 xylanases, which might be crucial for creating an additional aglycone subsite (+3) of the catalytic site. Finally, RcXyn30A activity is synergic to that of GH11 xylobiohydrolase.

Exploring a novel β-1,3-glucanosyltransglycosylase, MlGH17B, from a marine Muricauda lutaonensis strain for modification of laminari-oligosaccharides.

The marine environment, contains plentiful renewable resources, e.g. macroalgae with unique polysaccharides, motivating search for enzymes from marine microorganisms to explore conversion possibilities of the polysaccharides. In this study, the first GH17 glucanosyltransglycosylase, MlGH17B, from a marine bacterium (Muricauda lutaonensis), was characterized. The enzyme was moderately thermostable with Tm at 64.4°C and 73.2°C, but an activity optimum at 20°C, indicating temperature sensitive active site interactions. MlGH17B uses β-1,3 laminari-oligosaccharides with a degree of polymerization (DP) of 4 or higher as donors. Two glucose moieties (bound in the aglycone +1 and +2 subsites) are cleaved off from the reducing end of the donor while the remaining part (bound in the glycone subsites) is transferred to an incoming β-1,3 glucan acceptor, making a β-1,6-linkage, thereby synthesizing branched or kinked oligosaccharides. Synthesized oligosaccharides up to DP26 were detected by mass spectrometry analysis, showing that repeated transfer reactions occurred, resulting in several β-1,6-linked branches. The modeled structure revealed an active site comprising five subsites: three glycone (-3, -2 and -1) and two aglycone (+1 and +2) subsites, with significant conservation of substrate interactions compared to the only crystallized 1,3-β-glucanosyltransferase from GH17 (RmBgt17A from the compost thriving fungus Rhizomucor miehei), suggesting a common catalytic mechanism, despite different phylogenetic origin, growth environment, and natural substrate. Both enzymes lacked the subdomain extending the aglycone subsites, found in GH17 endo-β-glucanases from plants, but this extension was also missing in bacterial endoglucanases (modeled here), showing that this feature does not distinguish transglycosylation from hydrolysis, but may rather relate to phylogeny.

Molecular dynamics simulation guided distal mutation of Thermotoga naphthophila β-glucosidase for significantly enhanced synthesis of galactooligosaccharides and expanded product scope

For efficient enzymatic production of health-beneficial galactooligosaccharides (GOSs), a glycone (−1)/aglycone (+2) subsite mutation strategy to engineer a thermophilic GH1 β-glucosidase (Tn0602) from Thermotoga naphthophila RKU-10 was introduced. Six single mutation variants (F226G, N246G, N246E, N222F, N222Y, G224T) and two double mutants (F226GF414S, F226GF414Y) were designed. The +2-subsite variant F226G produced 136 mM galactooligosaccharide 1.2-fold more than the wild type (115 mM). More significantly, a superimposed mutation of the −1/+2 subsites F226G/F414S gave a total GOS production of 314 mM (82.16% lactose conversion), 2.7-fold higher than the total GOS production of the wild type. Furthermore, the variant F226GF414S was profiled 241 mM of trisaccharide (galβ (1 → 3)/(1 → 4) lactose) and 73 mM tetrasaccharide (galβ (1 → 3)/(1 → 4) galβ (1 → 3)/(1 → 4) lactose). According to a 300-ns molecular dynamic simulation, the superimposed mutation increased GOS productivity and expanded the scope of products by changing the structural flexibility and reducing the steric hindrance of the substrate tunnel. Overall, our study successfully demonstrated that a − 1/+2 subsite mutagenesis method could be used in β-glucosidases Tn0602 to improve enzyme productivity and expand product scope, which could be a potential route to evolve retaining glycosidases towards the desired direction.

A molecular dynamics simulations study of the ionic liquid effect on the β-glucosidase active site interactions with a flavonoid glycoside

Since the ionic liquid (IL) media is an appropriate environment for the enzymatic extraction of glycosides from natural resources, a detailed molecular understanding of the effect of IL concentration on enzyme function, with a focus on enzyme-substrate interactions, is critical. The best docking pose of Quercetin-4′-O-beta-D-glucopyranoside (Q-4′) in the active site of the β-glucosidase (BGL) enzyme was determined using molecular docking methods. Since flavonoid glycosides and enzymes are soluble in imidazolium-based ILs, the aqueous 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]) was used as a solvent in this study. The interactive residues of the BGL active site, as well as the types of interactions, in BGL–Q-4′ complex were then discovered using structural interaction fingerprints (SIFt) calculations. After that, the molecular perspective of the ionic liquid (IL) concentration effect on this complex and its interactions were simulated using 100 ns all-atom MD simulations. According to SIFt changes, changing the IL concentration influenced the overall interactions in the BGL–Q-4′ complex and involved residues. The RMSF findings indicated that the maximum flexibility of the interactive residues was observed in the 50% IL solution, however, flexibility changes were not high enough to alter enzyme activity. More analysis showed that solvent effects influenced hydrogen bond (HB) interactions with the substrate in both active site subsites; glycone (subsite -1) and aglycone subsites. Aromatic interactions followed a similar trend, suggesting that changes in IL concentration affected them. The solvent effects had a greater impact on the interactions with the aglycone part in both cases, which was attributed to aglycone subsite's greater solvent accessibility. Furthermore, it was found that IL concentration had a negative impact on the enzymatic hydrolysis process by influencing the formation of HB with Glu193 as well as the nucleophilic attack of Glu402. Finally, SDF findings indicated that an accumulation of IL molecules at the active site entrance could reduce enzyme activity by reducing the active site's accessibility to the substrate. By providing a molecular perspective on the BGL-substrate complex behavior in the IL system, our findings lead to a deeper understanding of the flavonoid glycoside hydrolysis process in these useful solvents.

Microwave-assisted xylanase reaction: impact in the production of prebiotic xylooligosaccharides.

The enzymatic production of prebiotic xylooligosaccharides (XOS) has become an attractive way to valorise lignocellulosic biomass. However, despite numerous xylanases reported for potential use in the production of XOS, most of the family GH10 also produce xylose. This monosaccharide can negatively affect the selectivity to stimulate the growth of intestinal microorganisms beneficial to human health. In this work, thermostable alkali-tolerant xylanase (BhXyn10A) from Bacillus halodurans S7 has been used to produce XOS under conventional convective heat transfer and microwave radiation. The microwave-assisted reaction markedly decreases the xylose content in the hydrolysates and significantly increases the yield of XOS, compared to conventional heating. Molecular dynamics simulations of BhXyn10A have shown increased fluctuations of the amino acids of the aglycone subsites suggesting that these subsites can determine the production of xylose. Thus, microwave heating could affect the amino acid fluctuations in the aglycone subsites reducing the xylose formation. These findings open up new avenues in enzyme technology for the production of XOS.

Understanding the Positional Binding and Substrate Interaction of a Highly Thermostable GH10 Xylanase from Thermotoga maritima by Molecular Docking.

Glycoside hydrolase family 10 (GH10) xylanases are responsible for enzymatic cleavage of the internal glycosidic linkages of the xylan backbone, to generate xylooligosaccharides (XOS) and xyloses. The topologies of active-site cleft determine the substrate preferences and product profiles of xylanases. In this study, positional bindings and substrate interactions of TmxB, one of the most thermostable xylanases characterized from Thermotoga maritima to date, was investigated by docking simulations. XOS with backbone lengths of two to five (X2–X5) were docked into the active-site cleft of TmxB by AutoDock The modeled complex structures provided a series of snapshots of the interactions between XOS and TmxB. Changes in binding energy with the length of the XOS backbone indicated the existence of four effective subsites in TmxB. The interaction patterns at subsites −2 to +1 in TmxB were conserved among GH10 xylanases whereas those at distal aglycone subsite +2, consisting of the hydrogen bond network, was unique for TmxB. This work helps in obtaining an in-depth understanding of the substrate-binding property of TmxB and provides a basis for rational design of mutants with desired product profiles.

Exploring the aglycone subsite of a GH11 xylanase for the synthesis of xylosides by transglycosylation reactions

Xylanases Tx-xyn10 and Tx-xyn11 were compared for their transxylosylation abilities in the presence of various acceptors. Tx-xyn10 exhibited a broad specificity for various acceptors, whereas xylanase Tx-xyn11 catalysed transxylosylation reactions only in presence of polyphenolic acceptors. A modelling approach was developed to study the molecular bottlenecks into the active site of the enzyme that could be responsible for this restricted specificity. The glycosyl-enzyme intermediate of Tx-xyn11 was modelled, and a rotamer of the Y78 residue was integrated. In silico mutations of some residues from the (+1) and (+2) subsites were tested for the deglycosylation step in the presence of non-polyphenolic acceptors. The results indicated that the mutant W126A was able to use aliphatic alcohols and benzyl alcohol as acceptors for transxylosylation. Experimental validation was tested by mutating the xylanase Tx-xyn11 at position W126 into alanine. The specific activity and catalytic efficiency of the W126A mutant during the hydrolysis of xylans decreased by 2-fold and 4-fold, respectively, compared to wild-type xylanase. Among tested acceptors, transxylosylation catalysed by mutant W126A was improved with benzyl alcohol leading to a 2-fold higher concentration of benzyl xylobioside, as predicted by in silico mutation. This improved transxylosylation in the presence of benzyl alcohol leading to higher synthesis of benzyl xylobioside could likely be explained by lowest steric hindrance in the aglycone subsite of the mutated xylanase. No secondary hydrolysis of benzyl xylobioside occurred for both wild-type and mutant xylanases. Finally, our results demonstrated that the modelling approach was limited and that accounting for protein dynamics can lead to improved models.

Structural insights of RmXyn10A – A prebiotic-producing GH10 xylanase with a non-conserved aglycone binding region

Hydrolysis of arabinoxylan (AX) by glycoside hydrolase family 10 (GH10) xylanases produces xylo- and arabinoxylo-oligosaccharides ((A)XOS) which have shown prebiotic effects. The thermostable GH10 xylanase RmXyn10A has shown great potential to produce (A)XOS. In this study, the structure of RmXyn10A was investigated, the catalytic module by homology modelling and site-directed mutagenesis and the arrangement of its five domains by small-angle X-ray scattering (SAXS). Substrate specificity was explored in silico by manual docking and molecular dynamic simulations. It has been shown in the literature that the glycone subsites of GH10 xylanases are well conserved and our results suggest that RmXyn10A is no exception. The aglycone subsites are less investigated, and the modelled structure of RmXyn10A suggests that loop β6α6 in the aglycone part of the active site contains a non-conserved α-helix, which blocks the otherwise conserved space of subsite +2. This structural feature has only been observed for one other GH10 xylanase. In RmXyn10A, docking revealed two alternative binding regions, one on either side of the α-helix. However, only one was able to accommodate arabinose-substitutions and the mutation study suggests that the same region is responsible for binding XOS. Several non-conserved structural features are most likely to be responsible for providing affinity for arabinose-substitutions in subsites +1 and +2. The SAXS rigid model of the modular arrangement of RmXyn10A displays the catalytic module close to the cell-anchoring domain while the carbohydrate binding modules are further away, likely explaining the observed lack of contribution of the CBMs to activity.

Arabinoxylanase from glycoside hydrolase family 5 is a selective enzyme for production of specific arabinoxylooligosaccharides

An arabinose specific xylanase from glycoside hydrolase family 5 (GH5) was used to hydrolyse wheat and rye arabinoxylan, and the product profile showed that it produced arabinose substituted oligosaccharides (AXOS) having 2–10 xylose residues in the main chain but no unsubstituted xylooligosaccharides (XOS). Molecular modelling showed that the active site has an open structure and that the hydroxyl groups of all xylose residues in the active site are solvent exposed, indicating that arabinose substituents can be accommodated in the glycone as well as the aglycone subsites. The arabinoxylan hydrolysates obtained with the GH5 enzyme stimulated growth of Bifidobacterium adolescentis but not of Lactobacillus brevis. This arabinoxylanase is thus a good tool for the production of selective prebiotics.

Engineering a thermostable Halothermothrix orenii β-glucosidase for improved galacto-oligosaccharide synthesis.

Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining β-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a β-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (−1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent kcat, but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower kcat). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with ~10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs.

Crystal structure of thermophilic dextranase from Thermoanaerobacter pseudethanolicus.

The crystal structures of the wild type and catalytic mutant Asp-312→Gly in complex with isomaltohexaose of endo-1,6-dextranase from the thermophilic bacterium Thermoanaerobacter pseudethanolicus (TpDex), belonging to the glycoside hydrolase family 66, were determined. TpDex consists of three structural domains, a catalytic domain comprising an (β/α)8-barrel and two β-domains located at both N- and C-terminal ends. The isomaltohexaose-complex structure demonstrated that the isomaltohexaose molecule was bound across the catalytic site, showing that TpDex had six subsites (-4 to +2) in the catalytic cleft. Marked movement of the Trp-376 side-chain along with loop 6, which was the side wall component of the cleft at subsite +1, was observed to occupy subsite +1, indicating that it might expel the cleaved aglycone subsite after the hydrolysis reaction. Structural comparison with other mesophilic enzymes indicated that several structural features of TpDex, loop deletion, salt bridge and surface-exposed charged residue, may contribute to thermostability.

Improved Transferase/Hydrolase Ratio through Rational Design of a Family 1 β-Glucosidase from Thermotoga neapolitana

Alkyl glycosides are attractive surfactants because of their high surface activity and good biodegradability and can be produced from renewable resources. Through enzymatic catalysis, one can obtain well-defined alkyl glycosides, something that is very difficult to do using conventional chemistry. However, there is a need for better enzymes to get a commercially feasible process. A thermostable β-glucosidase from the well-studied glycoside hydrolase family 1 from Thermotoga neapolitana, TnBgl1A, was mutated in an attempt to improve its value for synthesis of alkyl glycosides. This was done by rational design using prior knowledge from structural homologues together with a recently generated model of the enzyme in question. Three out of four studied mutations increased the hydrolytic reaction rate in an aqueous environment, while none displayed this property in the presence of an alcohol acceptor. This shows that even if the enzyme resides in a separate aqueous phase, the presence of an organic solvent has a great influence. We could also show that a single amino acid replacement in a less studied part of the aglycone subsite, N220F, improves the specificity for transglycosylation 7-fold and thereby increases the potential yield of alkyl glycoside from 17% to 58%.

Endo-β-D-1,4-mannanase fromChrysonilia sitophiladisplays a novel loop arrangement for substrate selectivity

The crystal structure of wild-type endo-β-D-1,4-mannanase (EC 3.2.1.78) from the ascomycete Chrysonilia sitophila (CsMan5) has been solved at 1.40 Å resolution. The enzyme isolated directly from the source shows mixed activity as both an endo-glucanase and an endo-mannanase. CsMan5 adopts the (β/α)(8)-barrel fold that is well conserved within the GH5 family and has highest sequence and structural homology to the GH5 endo-mannanases. Superimposition with proteins of this family shows a unique structural arrangement of three surface loops of CsMan5 that stretch over the active centre, promoting an altered topography of the binding cleft. The most relevant feature results from the repositioning of a long loop at the extremity of the binding cleft, resulting in a shortened glycone-binding region with two subsites. The other two extended loops flanking the binding groove produce a narrower cleft compared with the wide architecture observed in GH5 homologues. Two aglycone subsites (+1 and +2) are identified and a nonconserved tryptophan (Trp271) at the +1 subsite may offer steric hindrance. Taken together, these findings suggest that the discrimination of mannan substrates is achieved through modified loop length and structure.

Crystallographic analysis shows substrate binding at the −3 to +1 active-site subsites and at the surface of glycoside hydrolase family 11 endo-1,4-β-xylanases

GH 11 (glycoside hydrolase family 11) xylanases are predominant enzymes in the hydrolysis of heteroxylan, an abundant structural polysaccharide in the plant cell wall. To gain more insight into the protein-ligand interactions of the glycone as well as the aglycone subsites of these enzymes, catalytically incompetent mutants of the Bacillus subtilis and Aspergillus niger xylanases were crystallized, soaked with xylo-oligosaccharides and subjected to X-ray analysis. For both xylanases, there was clear density for xylose residues in the -1 and -2 subsites. In addition, for the B. subtilis xylanase, there was also density for xylose residues in the -3 and +1 subsite showing the spanning of the -1/+1 subsites. These results, together with the observation that some residues in the aglycone subsites clearly adopt a different conformation upon substrate binding, allowed us to identify the residues important for substrate binding in the aglycone subsites. In addition to substrate binding in the active site of the enzymes, the existence of an unproductive second ligand-binding site located on the surface of both the B. subtilis and A. niger xylanases was observed. This extra binding site may have a function similar to the separate carbohydrate-binding modules of other glycoside hydrolase families.

Understanding the Structural Basis for Substrate and Inhibitor Recognition in Eukaryotic GH11 Xylanases

Endo-β1,4-xylanases (xylanases) hydrolyse the β1,4 glycosidic bonds in the backbone of xylan. Although xylanases from glycoside hydrolase family 11 (GH11) have been extensively studied, several issues remain unresolved. Thus, the mechanism by which these enzymes hydrolyse decorated xylans is unclear and the structural basis for the variation in catalytic activity within this family is unknown. Furthermore, the mechanism for the differences in the inhibition of fungal GH11 enzymes by the wheat protein XIP-I remains opaque. To address these issues we report the crystal structure and biochemical properties of the Neocallimastix patriciarum xylanase NpXyn11A, which displays unusually high catalytic activity and is one of the few fungal GH11 proteins not inhibited by XIP-I. Although the structure of NpXyn11A could not be determined in complex with substrates, we have been able to investigate how GH11 enzymes hydrolyse decorated substrates by solving the crystal structure of a second GH11 xylanase, EnXyn11A (encoded by an environmental DNA sample), bound to ferulic acid-1,5-arabinofuranose-α1,3-xylotriose (FAX 3). The crystal structure of the EnXyn11A–FAX 3 complex shows that solvent exposure of the backbone xylose O2 and O3 groups at subsites −3 and +2 allow accommodation of α1,2-linked 4-methyl- D-glucuronic acid and L-arabinofuranose side chains. Furthermore, the ferulated arabinofuranose side chain makes hydrogen bonds and hydrophobic interactions at the +2 subsite, indicating that the decoration may represent a specificity determinant at this aglycone subsite. The structure of NpXyn11A reveals potential −3 and +3 subsites that are kinetically significant. The extended substrate-binding cleft of NpXyn11A, compared to other GH11 xylanases, may explain why the Neocallimastix enzyme displays unusually high catalytic activity. Finally, the crystal structure of NpXyn11A shows that the resistance of the enzyme to XIP-I is not due solely to insertions in the loop connecting β strands 11 and 12, as suggested previously, but is highly complex.

Determination of thioxylo‐oligosaccharide binding to family 11 xylanases using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and X‐ray crystallography

Noncovalent binding of thioxylo-oligosaccharide inhibitors, methyl 4-thio-alpha-xylobioside (S-Xyl2-Me), methyl 4,4II-dithio-alpha-xylotrioside (S-Xyl3-Me), methyl 4,4II,4III-trithio-alpha-xylotetroside (S-Xyl4-Me), and methyl 4,4II,4III,4IV-tetrathio-alpha-xylopentoside (S-Xyl5-Me), to three family 11 endo-1,4-beta-xylanases from Trichoderma reesei (TRX I and TRX II) and Chaetomium thermophilum (CTX) was characterized using electrospray ionization Fourier transform ion cyclotron resonance (FT-ICR) MS and X-ray crystallography. Ultra-high mass-resolving power and mass accuracy inherent to FT-ICR allowed mass measurements for noncovalent complexes to within |DeltaM|average of 2 p.p.m. The binding constants determined by MS titration experiments were in the range 10(4)-10(3) M-1, decreasing in the series of S-Xyl5-Me>or=S-Xyl4-Me>S-Xyl3-Me. In contrast, S-Xyl2-Me did not bind to any xylanase at the initial concentration of 5-200 microM, indicating increasing affinity with increasing number of xylopyranosyl units, with a minimum requirement of three. The crystal structures of CTX-inhibitor complexes gave interesting insights into the binding. Surprisingly, none of the inhibitors occupied any of the aglycone subsites of the active site. The binding to only the glycone subsites is nonproductive for catalysis, and yet this has also been observed for other family 11 xylanases in complex with beta-d-xylotetraose [Wakarchuk WW, Campbell RL, Sung WL, Davoodi J & Makoto Y (1994) Protein Sci3, 465-475, and Sabini E, Wilson KS, Danielsen S, Schulein M & Davies GJ (2001) Acta CrystallogrD57, 1344-1347]. Therefore, the role of the aglycone subsites remains controversial despite their obvious contribution to catalysis.

Directed Evolution of a Glycosynthase from Agrobacterium sp. Increases Its Catalytic Activity Dramatically and Expands Its Substrate Repertoire

The Agrobacterium sp. beta-glucosidase (Abg) is a retaining beta-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using alpha-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (kcat/Km) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using alpha-D-galactopyranosyl fluoride and 4-nitrophenyl beta-D-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (+1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6.

Influence of the aglycone region of the substrate binding cleft of Pseudomonas xylanase 10A on catalysis.

Pseudomonas cellulosa xylanase 10A (Pc Xyn10A) contains an extended substrate binding cleft comprising three glycone (-1 to -3) and four aglycone (+1 to +4) subsites and, typical of retaining glycoside hydrolases, exhibits transglycosylation activity at elevated substrate concentrations. In a previous study [Charnock, S. J., et al. (1997) J. Biol. Chem. 272, 2942-2951], it was demonstrated that the -2 subsite mutations E43A and N44A caused a 100-fold reduction in activity against xylooligosaccharides, but did not influence xylanase activity. This led to the proposal that the low activity of these mutants against xylooligosaccharides was due to nonproductive complex formation between these small substrates and the extended aglycone region of the active site. To test this hypothesis, key residues at the +2 (Asn182), +3 (Tyr255), and +4 (Tyr220) subsites were substituted for alanine, and the activity of the mutants against polysaccharides and oligosaccharides was evaluated. All the aglycone mutants exhibited greatly reduced or no transglycosylating activity, and the triple mutants, E43A/Y220A/Y255A and E43A/N182A/Y255A, had activity against xylotriose similar to that of E43A. The aglycone mutations caused an increase in both k(cat) and K(m) against xylan, with N182A/Y220A/Y255A and N182A/Y255A exhibiting 25- and 15-fold higher k(cat) values, respectively, than wild-type Pc Xyn10A. These data indicate that Glu43 plays a role in binding xylooligosaccharides, but not xylan, suggesting that the mechanisms by which Pc Xyn10A binds polysaccharides and oligosaccharides are distinct. The increased k(cat) of the mutants against xylan indicates that the aglycone region of wild-type Pc Xyn10A restricts the rate of catalysis by limiting diffusion of the cleaved substrate, generated at the completion of the k(2) step, out of the active site.