Structural characterization of the thermostableBradyrhizobium japonicumD-sorbitol dehydrogenase

Abstract

Bradyrhizobium japonicumsorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is D-glucitol (a synonym for D-sorbitol), although L-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Å resolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with D-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, theTmfor the orthologue fromRhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous β-sheet is formed between two monomers in the tetramer of theB. japonicumenzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio.