PNNL

Abstract

Beta-hydroxyacid dehydrogenase (ß-HAD) genes have been identified in all sequenced genomes of eukaryotes and prokaryotes. Their gene products catalyze the NAD+- or NADP+-dependent oxidation of various ß-hydroxy acid substrates into their corresponding semialdehyde. In many fungal and bacterial genomes, multiple ß-HAD genes are observed leading to the hypothesis that these gene products may have unique, uncharacterized metabolic roles specific to their species. The genomes of Geobacter sulfurreducens and Geobacter metallireducens each contain two potential ß-HAD genes. The protein sequences of one pair of these genes, Gs-ßHAD (Q74DE4) and Gm-ßHAD (Q39R98), have 65% sequence identity and 77% sequence similarity with each other. Both proteins reduce succinic semialdehyde, a metabolite of the GABA shunt. To further explore the structural and functional characteristics of these two ß-HADs with a potentially unique substrate specificity, crystal structures for Gs-ßHAD and Gm-ßHAD in complex with NADP+ were determined to a resolution of 1.89 Å and 2.07 Å, respectively. The structure of both proteins are similar, composed of 14 a-helices and nine ß-strands organized into two domains. Domain One (1-165) adopts a typical Rossmann fold composed of two a/ß units: a six-strand parallel ß-sheet surrounded by six a-helices (a1 – a6) followed by a mixed three-strand ß-sheet surrounded by two a-helices (a7 and a8). Domain Two (166-287) is composed of a bundle of seven a-helices (a9 – a14). Four functional regions conserved in all ß-HADs are spatially located near each other at the interdomain cleft in both Gs-ßHAD and Gm-ßHAD with a buried molecule of NADP+. The structural features of Gs-ßHAD and Gm-ßHAD are described in relation to the four conserved consensus sequences characteristic of ß-HADs and the potential biochemical importance of these enzymes as an alternative pathway for the degradation of succinic semialdehyde.