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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Bioproducts Research » Research » Publications at this Location » Publication #350422
Title: Biochemical characterization of caulobacter crescentus xylose dehydrogenase

Author
item Lee, Charles
item JORDAN, DOUGLASS - Retired ARS Employee
item STOLLER, ROSE - Retired ARS Employee
item Kibblewhite, Rena
item Wagschal, Kurt

Submitted to: International Journal of Biological Macromolecules
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/25/2018
Publication Date: 6/26/2018
Citation: Lee, C.C., Jordan, D.B., Stoller, R.J., Kibblewhite, R.E., Wagschal, K.C. 2018. Biochemical characterization of caulobacter crescentus xylose dehydrogenase. International Journal of Biological Macromolecules. 118:1362-1367. https://doi.org/10.1016/j.ijbiomac.2018.06.124.
DOI: https://doi.org/10.1016/j.ijbiomac.2018.06.124

Interpretive Summary: Waste lignocelluosic biomass from agricultural residues represents a tremendous source for chemical feedstocks and fuels. Valorization of all the components of lignocellulosic biomass is an essential step in fully leveraging this resource. Xylose sugar is a common component of hemicellulose, the second largest fraction of biomass. Many groups have developed biological conversions of xylose to value-added products by recombinant expression of the xylose dehydrogenase enzyme from Caulobacter crescentus. A detailed understanding of the mechanism of this enzyme would be invaluable in engineering more efficient versions. Therefore, we have produced and purified a recombinant version of this enzyme in order to conduct kinetic studies including both the forward and reverse physiological reactions of this enzyme. We demonstrate how the enzyme binds its critical cofactors (NAD+ or NADH), and we determine that dissociations of the sugar products from the enzyme complexes are the major rate limiting steps in both directions.

Technical Abstract: Valorization of all the components of lignocellulosic biomass is an essential step in minimizing our reliance on products sourced from non-renewable feedstocks. 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.