Identification, functional characterization, and structural analysis of an atypical l-threonate 3-dehydrogenase.

Diverse bacteria possess unusual gene clusters containing cryptic genes of unknown function, which are often related to the metabolism of sugars and sugar acids. In 1964, Aspen and Jakoby first isolated and characterized an NAD+-dependent l-threonate 3-dehydrogenase (Ltn3D; Enzyme Commission 1.1.1.129) from Pseudomonas sp. (J Biol Chem 239, 710-713), the molecular identity of which has remained unknown for over 60 years. Here, we have utilized bacterial genome context, together with biochemical and structural characterization, to reveal that GL300_RS07945 in Paracoccus litorisediminis encodes a representative NADP+-preferring Ltn3D. The crystal structure of the Michaelis ternary complex indicated that this enzyme is a member of the short-chain dehydrogenases/reductase superfamily, yet it differed in the recognition of the 2'-phosphate group of NADP+ between two adjacent arginine residues (Arg33 and Arg34). The C-3 atom of the competitive inhibitor tartronate was rationally positioned in close proximity to the nicotinamide ring for the catalysis. The reaction catalyzed by Ltn3D constitutes a distinct bypass route for the direct conversion of l-threonate to 3-oxo-l-threonate, which differs from the known sequential steps involving a dehydrogenase (l-threonate 2-dehydrogenase) and an isomerase (OtnI). In contrast to l-threonate 2-dehydrogenase, Ltn3D efficiently oxidized the 3-OH of homologous five- and six-carbon sugar acids, in addition to l-threonate. Among them, d-gluconate, potentially produced by GL300_RS07940 as a bifunctional 2-keto-d-gluconate/2-keto-l-gluconate reductase, could be converted to d-ribulose 5-phosphate by Ltn3D, followed by the action of a kinase (3OtnK) and a decarboxylase (3OtnC) in vitro. Altogether, our data suggest that Ltn3D constitutes a unique evolutionary innovation for the catabolism of four- to six-carbon sugar acids.