Cloning, expression, purification, crystallization and X-ray crystallographic analysis of glucuronic acid dehydrogenase from Chromohalobacter salexigens

Abstract

Glucuronic acid dehydrogenase (GluUADH), the product of the Csal-2474 gene from the halophilic bacterium Chromohalobacter salexigens DSM 3043, is an enzyme with potential use in the conversion of glucuronic acid in seaweed biomass to fuels and chemicals. GluUADH is an enzyme that catalyzes the oxidation of glucuronic acid (GluUA) and galacturonic acid (GalUA) and has a preference for NAD(+) rather than NADP(+) as a cofactor. Recombinant GluUADH was crystallized in the presence of 0.2 Mcalcium acetate, 0.1 M Tris-HCl pH 7.0 and 20% PEG 3000 at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.1 angstrom. The GluUADH crystal belonged to space group P6(3), with unit-cell parameters a = b = 122.58, c = 150.49 angstrom, gamma = 120 degrees. With one molecule per asymmetric unit, the crystal volume per unit protein weight (V-M) is 2.78 angstrom(3) Da(-1). The structure was solved by the single anomalous dispersion method and structure refinement is in progress.