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.

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.

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.

Biochemical Characterization of a GH43 ß-Xylosidase from Bacteroides ovatus.

Divalent metal-activated glycoside hydrolase family 43 (GH43) ß-xylosidases have been found to have high k cat/K m for xylooligosaccharides and may demonstrate high efficacy in industrial reactors digesting hemicellulose. By searching an amino acid database, we found a Bacteroides ovatus GH43 ß-xylosidase termed BoXA that is 81% identical in overall amino acid sequence to a GH43, divalent metal-activated ß-xylosidase with high k cat/K m, and also it has 19 of 20 residues in the active site conserved. However, unlike its metal-activated homolog, the B. ovatus enzyme does not lose activity after extensive EDTA treatment nor does it gain activity by addition of divalent metal ions. Thus, either it cannot be activated by divalent metal or it maintains a tightly bound, non-exchangeable metal ion. At 25 °C and pH 6.0, the k cat is 69 s-1 for xylobiose and k cat/K m is 210 s-1 mM-1 for xylotriose, with the latter being 0.7 that of the highest known value. The determined K i for D-glucose is 4.9 M, which is the highest known for a ß-xylosidase. The enzyme has potential utility operating in bioreactors digesting plant biomass.

Isolation and divalent-metal activation of a ß-xylosidase, RUM630-BX.

The gene encoding RUM630-BX, a ß-xylosidase/arabinofuranosidase, was identified from activity-based screening of a cow rumen metagenomic library. The recombinant enzyme is activated as much as 14-fold (kcat) by divalent metals Mg(2+), Mn(2+) and Co(2+) but not by Ca(2+), Ni(2+), and Zn(2+). Activation of RUM630-BX by Mg(2+) (t0.5 144 s) is slowed two-fold by prior incubation with substrate, consistent with the X-ray structure of closely related xylosidase RS223-BX that shows the divalent-metal activator is at the back of the active-site pocket so that bound substrate could block its entrance. The enzyme is considerably more active on natural substrates than artificial substrates, with activity (kcat/Km) of 299 s(-1) mM(-1) on xylotetraose being the highest reported.