A general correction to catalytic rates determined for nonprocessive exo-depolymerases acting on both substrate and product in the initial-rate measurement.

We recently reported on the kinetics of the polygalacturonase TtGH28 acting on trimer and dimer substrates. When the starting substrate for hydrolysis is the trimer, the product dimer is also subject to hydrolysis, resulting in discrepancies when either the concentration of dimer or monomer product is used for analysis of trimer hydrolysis. Here, we derive a method for determining catalytic rates of exo-hydrolases acting on trimer (and higher order) substrates when products may also be substrates for hydrolysis and show how this correction may be applied for TtGH28.

Rate-limiting steps of a stereochemistry retaining ß-d-xylosidase from Geobacillus stearothermophilus acting on four substrates.

Kinetic experiments of GSXynB2, a GH52 retaining ß-xylosidase, acting on 2-nitrophenyl-ß-d-xylopyranoside (2NPX), 4-nitrophenyl-ß-d-xylopyranoside (4NPX), 4-methylumbelliferyl-ß-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. kcat 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). kcat 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 pKa of 7.2) of the Brønsted plot pattern (logkcat vs pKa of phenol leaving group) is thus possibly explained by 4NP release. The pH dependency of kcat 2NPX encompasses 2 bell-shaped curves with peaks of pH 3 and pH 7.

Directed evolution of GH43 ß-xylosidase XylBH43 thermal stability and L186 saturation mutagenesis.

Directed evolution of ß-xylosidase XylBH43 using a single round of gene shuffling identified three mutations, R45K, M69P, and L186Y, that affect thermal stability parameter K(t)°·5 by -1.8 ± 0.1, 1.7 ± 0.3, and 3.2 ± 0.4 °C, respectively. In addition, a cluster of four mutations near hairpin loop-D83 improved K(t)°·5 by ~3 °C; none of the individual amino acid changes measurably affect K(t)°·5. Saturation mutagenesis of L186 identified the variant L186K as having the most improved K(t)°·5 value, by 8.1 ± 0.3 °C. The L186Y mutation was found to be additive, resulting in K(t)°·5 increasing by up to 8.8 ± 0.3 °C when several beneficial mutations were combined. While k cat of xylobiose and 4-nitrophenyl-ß-D-xylopyranoside were found to be depressed from 8 to 83 % in the thermally improved mutants, K(m), K(ss) (substrate inhibition), and K(i) (product inhibition) values generally increased, resulting in lessened substrate and xylose inhibition.

Rehabilitation of faulty kinetic determinations and misassigned glycoside hydrolase family of retaining mechanism ß-xylosidases.

We obtained Cx1 from a commercial supplier, whose catalog listed it as a ß-xylosidase of glycoside hydrolase family 43. NMR experiments indicate retention of anomeric configuration in its reaction stereochemistry, opposing the assignment of GH43, which follows an inverting mechanism. Partial protein sequencing indicates Cx1 is similar to but not identical to ß-xylosidases of GH52, including Q09LZ0, that have retaining mechanisms. Q09LZ0 ß-xylosidase had been characterized biochemically in kinetic reactions that contained Tris. We overproduced Q09LZ0 and demonstrated that Tris is a competitive inhibitor of the ß-xylosidase. Also, the previous work used grossly incorrect extinction coefficients for product 4-nitrophenol. We redetermined kinetic parameters using reactions that omitted Tris and using correct extinction coefficients for 4-nitrophenol. Cx1 and Q09LZ0 ß-xylosidases were thus shown to possess similar kinetic properties when acting on 4-nitrophenyl-ß-d-xylopyranoside and xylobiose. kcat pH profiles of Cx1 and Q09LZ0 acting on 4-nitrophenyl-ß-d-xylopyranoside and xylobiose have patterns containing two rate increases with increasing acidity, not reported before for glycoside hydrolases. The dexylosylation step of 4-nitrophenyl-ß-d-xylopyranoside hydrolysis mediated by Q09LZ0 is not rate determining for kcat(4NPX).

Activation of a GH43 ß-xylosidase by divalent metal cations: slow binding of divalent metal and high substrate specificity.

RS223-BX of glycoside hydrolase family 43 is a ß-d-xylosidase that is strongly activated (k(cat)/K(m) as much as 116-fold) by the addition of divalent metal cations, Ca(2+), Co(2+), Fe(2+), Mg(2+), Mn(2+) and Ni(2+). Slow activation by Mg(2+) was demonstrated (k(on) 0.013 s(-1) mM(-1), k(off) 0.008 s(-1)) at pH 7.0 and 25 °C. k(off) and k(on) values are independent of Mg(2+) concentration, but k(off) and k(on) are slower in the presence of increasing levels of substrate 4-nitrophenyl-ß-D-xylopyranoside. The kinetics strongly suggest that M(2+) binds to the enzyme rapidly, forming E M(2+), followed by slow isomerization to the activated enzyme, E* M(2+). Moderately high values of kcat (7-30 s(-1)) were found for M(2+)-activated RS223-BX acting on xylobiose (natural substrate) at pH 7.0 and 25 °C. Certain M(2+)-activated RS223-BX exhibit the highest reported values of k(cat)/K(m) of any ß-xylosidase acting on natural substrates: for example, at pH 7.0 and 25°C, xylobiose (Mn(2+), 190 s(-1) mM(-1)), xylotriose (Ca(2+), 150 s(-1) mM(-1)) and xylotetraose (Ca(2+), 260 s(-1) mM(-1)). There is potential for the enzyme to add value to industrial saccharification operations at low substrate and high d-glucose and high d-xylose concentrations.

Highly active ß-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates.

The hemicellulose xylan constitutes a major portion of plant biomass, a renewable feedstock available for conversion to biofuels and other bioproducts. ß-xylosidase operates in the deconstruction of the polysaccharide to fermentable sugars. Glycoside hydrolase family 43 is recognized as a source of highly active ß-xylosidases, some of which could have practical applications. The biochemical details of four GH43 ß-xylosidases (those from Alkaliphilus metalliredigens QYMF, Bacillus pumilus, Bacillus subtilis subsp. subtilis str. 168, and Lactobacillus brevis ATCC 367) are examined here. Sedimentation equilibrium experiments indicate that the quaternary states of three of the enzymes are mixtures of monomers and homodimers (B. pumilus) or mixtures of homodimers and homotetramers (B. subtilis and L. brevis). k cat and k cat/K m values of the four enzymes are higher for xylobiose than for xylotriose, suggesting that the enzyme active sites comprise two subsites, as has been demonstrated by the X-ray structures of other GH43 ß-xylosidases. The K i values for D-glucose (83.3-357 mM) and D-xylose (15.6-70.0 mM) of the four enzymes are moderately high. The four enzymes display good temperature (K t (0.5) ∼ 45 °C) and pH stabilities (>4.6 to <10.3). At pH 6.0 and 25 °C, the enzyme from L. brevis ATCC 367 displays the highest reported k cat and k cat/K m on natural substrates xylobiose (407 s(-1), 138 s(-1) mM(-1)), xylotriose (235 s(-1), 80.8 s(-1) mM(-1)), and xylotetraose (146 s(-1), 32.6 s(-1) mM(-1)).

Plant cell walls to ethanol.

Conversion of plant cell walls to ethanol constitutes second generation bioethanol production. The process consists of several steps: biomass selection/genetic modification, physiochemical pretreatment, enzymatic saccharification, fermentation and separation. Ultimately, it is desirable to combine as many of the biochemical steps as possible in a single organism to achieve CBP (consolidated bioprocessing). A commercially ready CBP organism is currently unreported. Production of second generation bioethanol is hindered by economics, particularly in the cost of pretreatment (including waste management and solvent recovery), the cost of saccharification enzymes (particularly exocellulases and endocellulases displaying kcat ~1 s-1 on crystalline cellulose), and the inefficiency of co-fermentation of 5- and 6-carbon monosaccharides (owing in part to redox cofactor imbalances in Saccharomyces cerevisiae).

Opposing influences by subsite -1 and subsite +1 residues on relative xylopyranosidase/arabinofuranosidase activities of bifunctional ß-D-xylosidase/α-L-arabinofuranosidase.

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-ß-d-xylopyranoside (4NPX)>xylobiose (X2). The bifunctional ß-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.

Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural.

An effective means of relieving the toxicity of furan aldehydes, furfural (FFA) and 5-hydroxymethylfurfural (HMF), on fermenting organisms is essential for achieving efficient fermentation of lignocellulosic biomass to ethanol and other products. Ari1p, an aldehyde reductase from Saccharomyces cerevisiae, has been shown to mitigate the toxicity of FFA and HMF by catalyzing the NADPH-dependent conversion to corresponding alcohols, furfuryl alcohol (FFOH) and 5-hydroxymethylfurfuryl alcohol (HMFOH). At pH 7.0 and 25°C, purified Ari1p catalyzes the NADPH-dependent reduction of substrates with the following values (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFA (23.3, 1.82, 12.8), HMF (4.08, 0.173, 23.6), and dl-glyceraldehyde (2.40, 0.0650, 37.0). When acting on HMF and dl-glyceraldehyde, the enzyme operates through an equilibrium ordered kinetic mechanism. In the physiological direction of the reaction, NADPH binds first and NADP(+) dissociates from the enzyme last, demonstrated by k(cat) of HMF and dl-glyceraldehyde that are independent of [NADPH] and (K(ia)(NADPH)/k(cat)) that extrapolate to zero at saturating HMF or dl-glyceraldehyde concentration. Microscopic kinetic parameters were determined for the HMF reaction (HMF+NADPH↔HMFOH+NADP(+)), by applying steady-state, presteady-state, kinetic isotope effects, and dynamic modeling methods. Release of products, HMFOH and NADP(+), is 84% rate limiting to k(cat) in the forward direction. Equilibrium constants, [NADP(+)][FFOH]/[NADPH][FFA][H(+)]=5600×10(7)M(-1) and [NADP(+)][HMFOH]/[NADPH][HMF][H(+)]=4200×10(7)M(-1), favor the physiological direction mirrored by the slowness of hydride transfer in the non-physiological direction, NADP(+)-dependent oxidation of alcohols (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFOH (0.221, 0.00158, 140) and HMFOH (0.0105, 0.000104, 101).

Engineering lower inhibitor affinities in ß-D-xylosidase of Selenomonas ruminantium by site-directed mutagenesis of Trp145.

ß-D-Xylosidase/α-L-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme reported for catalyzing hydrolysis of 1,4-ß-D-xylooligosaccharides to D-xylose. One property that could use improvement is its relatively high affinities for D-glucose and D-xylose (K (i) ~ 10 mM), which would impede its performance as a catalyst in the saccharification of lignocellulosic biomass for the production of biofuels and other value-added products. Previously, we discovered that the W145G variant expresses K(i)(D-glucose) and K(i)(D-xylose) twofold and threefold those of the wild-type enzyme. However, in comparison to the wild type, the variant expresses 11% lower k(cat)(D-xylobiose) and much lower stabilities to temperature and pH. Here, we performed saturation mutagenesis of W145 and discovered that the variants express K (i) values that are 1.5-2.7-fold (D-glucose) and 1.9-4.6-fold (D-xylose) those of wild-type enzyme. W145F, W145L, and W145Y express good stability and, respectively, 11, 6, and 1% higher k(cat)(D-xylobiose) than that of the wild type. At 0.1 M D-xylobiose and 0.1 M D-xylose, kinetic parameters indicate that W145F, W145L, and W145Y catalytic activities are respectively 46, 71, and 48% greater than that of the wild-type enzyme.

Aminoalcohols as probes of the two-subsite active site of beta-D-xylosidase from Selenomonas ruminantium.

Catalysis and inhibitor binding by the GH43 beta-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-beta-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-alpha-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.

Stereochemistry of furfural reduction by a Saccharomyces cerevisiae aldehyde reductase that contributes to in situ furfural detoxification.

Ari1p from Saccharomyces cerevisiae, recently identified as an intermediate-subclass short-chain dehydrogenase/reductase, contributes in situ to the detoxification of furfural. Furfural inhibits efficient ethanol production by yeast, particularly when the carbon source is acid-treated lignocellulose, which contains furfural at a relatively high concentration. NADPH is Ari1p's best known hydride donor. Here we report the stereochemistry of the hydride transfer step, determined by using (4R)-[4-(2)H]NADPD and (4S)-[4-(2)H]NADPD and unlabeled furfural in Ari1p-catalyzed reactions and following the deuterium atom into products 2-furanmethanol or NADP(+). Analysis of the products demonstrates unambiguously that Ari1p directs hydride transfer from the si face of NADPH to the re face of furfural. The singular orientation of substrates enables construction of a model of the Michaelis complex in the Ari1p active site. The model reveals hydrophobic residues near the furfural binding site that, upon mutation, may increase specificity for furfural and enhance enzyme performance. Using (4S)-[4-(2)H]NADPD and NADPH as substrates, primary deuterium kinetic isotope effects of 2.2 and 2.5 were determined for the steady-state parameters k(cat)(NADPH) and k(cat)/K(m)(NADPH), respectively, indicating that hydride transfer is partially rate limiting to catalysis.

Properties and applications of microbial beta-D-xylosidases featuring the catalytically efficient enzyme from Selenomonas ruminantium.

Xylan 1,4-beta-D-xylosidase catalyzes hydrolysis of non-reducing end xylose residues from xylooligosaccharides. The enzyme is currently used in combination with beta-xylanases in several large-scale processes for improving baking properties of bread dough, improving digestibility of animal feed, production of D-xylose for xylitol manufacture, and deinking of recycled paper. On a grander scale, the enzyme could find employment alongside cellulases and other hemicellulases in hydrolyzing lignocellulosic biomass so that reaction product monosaccharides can be fermented to biofuels such as ethanol and butanol. Catalytically efficient enzyme, performing under saccharification reactor conditions, is critical to the feasibility of enzymatic saccharification processes. This is particularly important for beta-xylosidase which would catalyze breakage of more glycosidic bonds of hemicellulose than any other hemicellulase. In this paper, we review applications and properties of the enzyme with emphasis on the catalytically efficient beta-D-xylosidase from Selenomonas ruminantium and its potential use in saccharification of lignocellulosic biomass for producing biofuels.

Engineering lower inhibitor affinities in beta-D-xylosidase.

Beta-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 (K (i) values of approximately 10(-2) M). Higher K (i)'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 degrees screen selects for activity (active/inactive screen) and the 2 degrees screen selects for a higher K (i(D-xylose)) and its subsequent use in screening approximately 5,900 members of an SXA enzyme library prepared using error-prone PCR. In one variant, termed SXA-C3, K (i(D-xylose)) is threefold and K (i(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 A from the active site, and its side chain contacts three active-site residues, two in subsite +1 and one in subsite -1.

Penicillium camemberti galacturonate reductase: C-1 oxidation/reduction of uronic acids and substrate inhibition mitigation by aldonic acids.

The enzyme galacturonate oxidoreductase PcGOR from Penicillium camemberti reduces the C-1 carbon of D-glucuronate and C-4 epimer D-galacturonate to their corresponding aldonic acids, important reactions in both pectin catabolism and ascorbate biosynthesis. PcGOR was active on both glucuronic acid and galacturonic acid, with similar substrate specificities (kcat/Km) using the preferred co-substrate NADPH. Substrate acceptance extended to lactone congeners, and D-glucurono-3,6-lactone was converted to L-gulono-1,4-lactone, an immediate precursor of ascorbate. Reaction with glucuronate showed only minor substrate inhibition, and the product L-gulonate and L-gulono-1,4-lactone were both found to be competitive inhibitors with Ki in the low mM range. In contrast, reaction with C-4 epimer galacturonate displayed marked substrate inhibition. Moreover, the product L-galactonate and L-galactono-1,4-lactone were observed to mitigate substrate inhibition by galacturonate, with the lactone having a greater effect than the acid.

Extracellular hemicellulolytic enzymes from the maize endophyte Acremonium zeae.

Microorganisms that colonize plants require a number of hydrolytic enzymes to help degrade the cell wall. The maize endophyte Acremonium zeae was surveyed for production of extracellular enzymes that hydrolyze cellulose and hemicellulose. The most prominent enzyme activity in cell-free culture medium from A. zeae NRRL 6415 was xylanase, with a specific activity of 60 U/mg from cultures grown on crude corn fiber. Zymogram analysis following SDS-PAGE indicated six functional xylanase polypeptides of the following masses: 51, 44, 34, 29, 23, and 20 kDa. Xylosidase (0.39 U/mg), arabinofuranosidase (1.2 U/mg), endoglucanase (2.3 U/mg), cellobiohydrolase (1.3 U/mg), and beta-glucosidase (0.85 U/mg) activities were also detected. Although apparently possessing a full complement of hemicellulolytic activities, cell-free culture supernatants prepared from A. zeae required an exogenously added xylosidase to release more than 90% of the xylose and 80% of the arabinose from corn cob and wheat arabinoxylans. The hydrolytic enzymes from A. zeae may be suitable for application in the bioconversion of lignocellulosic biomass into fermentable sugars.

Variation in relative substrate specificity of bifunctional beta-D-xylosidase/alpha-L-arabinofuranosidase by single-site mutations: roles of substrate distortion and recognition.

To probe differential control of substrate specificities for 4-nitrophenyl-alpha-l-arabinofuranoside (4NPA) and 4-nitrophenyl-beta-d-xylopyranoside (4NPX), residues of the glycone binding pocket of GH43 beta-d-xylosidase/alpha-l-arabinofuranosidase from Selenomonas ruminantium were individually mutated to alanine. Although their individual substrate specificities (kcat/Km)(4NPX) and (kcat/Km)(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 (kcat/Km)(4NPX)/(kcat/Km)(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 (kcat/Km)(4NPX) and (kcat/Km)(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.

Structure of the two-subsite beta-d-xylosidase from Selenomonas ruminantium in complex with 1,3-bis[tris(hydroxymethyl)methylamino]propane.

The three-dimensional structure of the catalytically efficient beta-xylosidase from Selenomonas ruminantium in complex with competitive inhibitor 1,3-bis[tris(hydroxymethyl)methylamino]propane (BTP) was determined by using X-ray crystallography (1.3A resolution). Most H bonds between inhibitor and protein occur within subsite -1, including one between the carboxyl group of E186 and an N group of BTP. The other N of BTP occupies subsite +1 near K99. E186 (pK(a) 7.2) serves as catalytic acid. The pH (6-10) profile for 1/K(i)((BTP)) is bell-shaped with pK(a)'s 6.8 and 7.8 on the acidic limb assigned to E186 and inhibitor groups and 9.9 on the basic limb assigned to inhibitor. Mutation K99A eliminates pK(a) 7.8, strongly suggesting that the BTP monocation binds to the dianionic enzyme D14(-)E186(-). A sedimentation equilibrium experiment estimates a K(d) ([dimer](2)/[tetramer]) of 7 x 10(-9)M. Similar k(cat) and k(cat)/K(m) values were determined when the tetramer/dimer ratio changes from 0.0028 to 26 suggesting that dimers and tetramers are equally active forms.

Absence or presence of metal ion activation in two structurally similar GH43 ß-xylosidases.

Two GH43 ß-xylosidases, RS223-BX from a rice straw metagenomic library, and BoXA from Bacteroides ovatus, that share similar amino acid sequences (81% identical) and 19 of 20 active-site residues, were compared by using site-directed mutagenesis of Asp and His residues implicated in metal binding. Thus, RS223-BX is strongly activated by divalent-metal cations and the previously published X-ray structure of this enzyme shows that a Ca2+ cation is chelated by an active-site Asp carboxyl group and an active-site His. Mutation to Ala causes 90% loss of activity for the Asp mutant and 98% loss of activity for the His mutant, indicating their importance to catalysis. For the other enzyme (BoXA), mutation to Ala causes 20% loss of activity for the His mutant and 40% gain of activity for the Asp mutant, indicating the lack of importance for activity of the native residues and the lack of metal-dependency, given that the Asp residue occupies the active site to secure the metal cation in known metal ion dependent GH43 xylosidases. The high activity of the BoXA mutants compared to that of the analogous RS223-BX mutants further undermines the possibility that BoXA maintains a tightly bound metal cofactor resistant to EDTA extraction. The results strengthen our conclusion that the very similar proteins differ in one being metal ion dependent and one not.

Biochemical characterization of Caulobacter crescentus xylose dehydrogenase.

d-Xylose sugar is a common component of hemicellulose, the second largest fraction of biomass. Many groups have developed biological conversions of d-xylose to value-added products by recombinant expression of the xylose dehydrogenase enzyme from Caulobacter crescentus. This enzyme uses NAD+ as a cofactor to oxidize d-xylose to d-xylono-1,4-lactone. A detailed understanding of the mechanism of this enzyme could be useful in engineering more efficient versions. Therefore, we have conducted kinetic studies including both the forward and reverse physiological reactions of this enzyme. We demonstrate that the enzyme's substrate binding mode follows a sequential steady state ordered mechanism with NAD+ or NADH binding first. Furthermore, the kcat of the reaction in the direction of NAD+ reduction is 10-fold higher than that of the reverse reaction. From rapid reaction studies, we demonstrate the binding of NAD+ and NADH to the free enzyme and that hydride transfer occurs in a fast step followed by a much slower steady state. We calculate that the dissociations of the sugar products from the enzyme complexes are the major rate limiting steps in both directions.