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 k_cat 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.