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Kinetic experiments of GSXynB2, a GH52 retaining β-xylosidase, acting on 2-nitrophenyl-β- d -xylopyranoside (2NPX), 4-nitrophenyl-β- d -xylopyranoside (4NPX), 4-methylumbelliferyl-β- d -xylopyranoside (MuX) and xylobiose (X2) were conducted at pH 7.0 and 25 °C. Catalysis proceeds in two steps (xylodidation followed by dexylosidation): E + substrate TO E-xylose + leaving group TO E + xylose. k cat falls into two groups: 4NPX (1.95 s −1 ) and 2NPX, MuX and X2 (15.8 s −1 , 12.6 s −1 , 12.8 s −1 , respectively). Dexylosylation (E-xylose to E + xylose), the common step for the enzymatic hydrolysis of the four substrates, must exceed 15.8 s −1 . k cat of 4NPX would seem mainly limited by xylosylation (step 1) and the other three substrates would seem mainly limited by dexylosylation (step 2) – a conclusion that critically lacks chemical justification (compare 4NPX and 2NPX). Presteady-state rates indicate rapid xylosidation rates for all substrates so a later step (not dexylosidation) is rate-limiting for 4NPX. That 2NPX is an onlier and 4NPX is an outlier (both leaving group p K a of 7.2) of the Brønsted plot pattern (log k cat vs p K a of phenol leaving group) is thus possibly explained by 4NP release. The pH dependency of k cat 2NPX encompasses 2 bell-shaped curves with peaks of pH 3 and pH 7. [ABSTRACT FROM AUTHOR]

Abstract: Conformational inversion occurs 7–8kcal/mol more readily in furanoses than pyranoses. This difference is exploited here to probe for active-site residues involved in distorting pyranosyl substrate toward reactivity. Spontaneous glycoside hydrolysis rates are ordered 4-nitrophenyl-α-l-arabinofuranoside (4NPA)>4-nitrophenyl-β-d-xylopyranoside (4NPX)>xylobiose (X2). The bifunctional β-d-xylosidase/α-l-arabinofuranosidase exhibits the opposite order of reactivity, illustrating that the enzyme is well equipped in using pyranosyl groups of natural substrate X2 in facilitating glycoside hydrolysis. Probing the roles of all 17 active-site residues by single-site mutation to alanine and by changing both moieties of substrate demonstrates that the mutations of subsite −1 residues decrease the ratio k cat 4NPX/4NPA, suggesting that the native residues support pyranosyl substrate distortion, whereas the mutations of subsite +1 and the subsite −1/+1 interface residues increase the ratio k cat 4NPX/4NPA, suggesting that the native residues support other factors, such as C1 migration and protonation of the leaving group. Alanine mutations of subsite −1 residues raise k cat X2/4NPX and alanine mutations of subsite +1 and interface residues lower k cat X2/4NPX. We propose that pyranosyl substrate distortion is supported entirely by native residues of subsite −1. Other factors leading to the transition state are supported entirely by native residues of subsite +1 and interface residues. [Copyright &y& Elsevier]

β- d-Xylosidase catalyzes hydrolysis of xylooligosaccharides to d-xylose residues. The enzyme, SXA from Selenomonas ruminantium, is the most active catalyst known for the reaction; however, its activity is inhibited by d-xylose and d-glucose ( Ki values of ∼10−2 M). Higher Ki’s could enhance enzyme performance in lignocellulose saccharification processes for bioethanol production. We report here the development of a two-tier high-throughput screen where the 1° screen selects for activity (active/inactive screen) and the 2° screen selects for a higher Ki( d-xylose) and its subsequent use in screening ∼5,900 members of an SXA enzyme library prepared using error-prone PCR. In one variant, termed SXA-C3, Ki( d-xylose) is threefold and Ki( d-glucose) is twofold that of wild-type SXA. C3 contains four amino acid mutations, and one of these, W145G, is responsible for most of the lost affinity for the monosaccharides. Experiments that probe the active site with ligands that bind only to subsite −1 or subsite +1 indicate that the changed affinity stems from changed affinity for d-xylose in subsite +1 and not in subsite −1 of the two-subsite active site. Trp145 is 6 Å from the active site, and its side chain contacts three active-site residues, two in subsite +1 and one in subsite −1. [ABSTRACT FROM AUTHOR]

Abstract: Catalysis and inhibitor binding by the GH43 β-xylosidase are governed by the protonation states of catalytic base (D14, pK a 5.0) and catalytic acid (E186, pK a 7.2) which reside in subsite −1 of the two-subsite active site. Cationic aminoalcohols are shown to bind exclusively to subsite −1 of the catalytically-inactive, dianionic enzyme (D14−E186−). Enzyme (E) and aminoalcohols (A) form E–A with the affinity progression: triethanolamine>diethanolamine>ethanolamine. E186A mutation raises the K i triethanolamine 1000-fold. By occupying subsite −1 with aminoalcohols, affinity of monosaccharide inhibitors (I) for subsite +1 is demonstrated. The single access route for ligands into the active site dictates ordered formation of E–A followed by E–A–I. E–A–I forms with the affinity progression: ethanolamine>diethanolamine>triethanolamine. The latter affinity progression is seen in formation of E–A–substrate complexes with substrate 4-nitrophenyl-β-d-xylopyranoside (4NPX). Inhibition patterns of aminoalcohols versus 4NPX appear competitive, noncompetitive, and uncompetitive depending on the strength of E–A–4NPX. E–A–substrate complexes form weakly with substrate 4-nitrophenyl-α-l-arabinofuranoside (4NPA), and inhibition patterns appear competitive. Biphasic inhibition by triethanolamine reveals minor (<0.03%) contamination of E186A preparations (including a His-Tagged form) by wild-type-like enzyme, likely originating from translational misreading. Aminoalcohols are useful in probing glycoside hydrolases; they cause artifacts when used unwarily as buffer components. [Copyright &y& Elsevier]

Abstract: To probe differential control of substrate specificities for 4-nitrophenyl-α-l-arabinofuranoside (4NPA) and 4-nitrophenyl-β-d-xylopyranoside (4NPX), residues of the glycone binding pocket of GH43 β-d-xylosidase/α-l-arabinofuranosidase from Selenomonas ruminantium were individually mutated to alanine. Although their individual substrate specificities (k cat/K m)4NPX and (k cat/K m)4NPA are lowered 330 to 280,000 fold, D14A, D127A, W73A, E186A, and H248A mutations maintain similar relative substrate specificities as wild-type enzyme. Relative substrate specificities (k cat/K m)4NPX/(k cat/K m)4NPA are lowered by R290A, F31A, and F508A mutations to 0.134, 0.407, and 4.51, respectively, from the wild type value of 12.3 with losses in (k cat/K m)4NPX and (k cat/K m)4NPA of 18 to 163000 fold. R290 and F31 reside above and below the C4 OH group of 4NPX and the C5 OH group of 4NPA, where they can serve as anchors for the two glycone moieties when their ring systems are distorted to transition-state geometries by raising the position of C1. Thus, whereas R290 and F31 provide catalytic power for hydrolysis of both substrates, the native residues are more important for 4NPX than 4NPA as the xylopyranose ring must undergo greater distortion than the arabinofuranose ring. F508 borders C4 and C5 of the two glycone moieties and can serve as a hydrophobic platform having more favorable interactions with xylose than arabinofuranose. [Copyright &y& Elsevier]

Abstract: The active site of the GH43 β-xylosidase from Selenomonas ruminantium comprises two subsites and a single access route for ligands. Steady-state kinetic experiments that included enzyme (E), inhibitory sugars (I and X) and substrate (S) establish examples of EI, EII, EIX, and EIS complexes. Protonation states of catalytic base (D14, pK a 5) and catalytic acid (E186, pK a 7) govern formation of inhibitor complexes and strength of binding constants: e.g., EII, EIX, and EIS occur only with the D14−E186H enzyme and d-xylose binds to D14−E186− better than to D14−E186H. Binding of two equivalents of l-arabinose to the D14−E186H enzyme is differentiated by the magnitude of equilibrium K i values (first binds tighter) and kinetically (first binds rapidly; second binds slowly). In applications, such as saccharification of herbaceous biomass for subsequent fermentation to biofuels, the highly efficient hydrolase can confront molar concentrations of sugars that diminish catalytic effectiveness by forming certain enzyme-inhibitor complexes. [Copyright &y& Elsevier]