title: Structural characterization of a β-hydroxyacid dehydrogenase from Geobacter sulfurreducens and Geobacter metallireducens with succinic semialdehyde reductase activity.
abstract: Beta-hydroxyacid dehydrogenase (β-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 β-hydroxy acid substrates into their corresponding semialdehyde. In many fungal and bacterial genomes, multiple β-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 β-HAD genes. The protein sequences of one pair of these genes, Gs-βHAD (Q74DE4) and Gm-βHAD (Q39R98), have 65% sequence identity and 77% sequence similarity with each other. Both proteins are observed to reduce succinic semialdehyde, a 4-carbon substrate instead of the typical β-HAD 3-carbon substrate, to γ-hydroxybutyric acid. To further explore the structural and functional characteristics of these two β-HADs with a less frequently observed substrate specificity, crystal structures for Gs-βHAD and Gm-βHAD in complex with NADP(+) were determined to a resolution of 1.89 Å and 2.07 Å, respectively. The structures of both proteins are similar, composed of 14 α-helices and nine β-strands organized into two domains. Domain 1 (1-165) adopts a typical Rossmann fold composed of two α/β units: a six-strand parallel β-sheet surrounded by six α-helices (α1-α6) followed by a mixed three-strand β-sheet surrounded by two α-helices (α7 and α8). Domain 2 (166-287) is composed of a bundle of seven α-helices (α9-α14). Four functional regions conserved in all β-HADs are spatially located near each other, with a buried molecule of NADP(+), at the interdomain cleft. Comparison of these Geobacter structures to a closely related β-HAD from Arabidopsis thaliana in the apo-NADP(+) and apo-substrate bound state suggests that NADP(+) binding effects a rigid body rotation between Domains 1 and 2. Bound near the Substrate-Binding and Catalysis Regions in two of the eight protomers in the asymmetric unit of Gm-βHAD is a glycerol molecule that may mimic features of bound biological substrates.
authors.affiliation: College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, People's Republic of China; Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA.
authors.forename: Yanfeng
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: Qin
authors.affiliation: College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, People's Republic of China. Electronic address: wshyyl@sina.com.
authors.forename: Shihua
authors.initials: S
authors.lastname: Wang
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA. Electronic address: garry.buchko@pnnl.gov.
authors.forename: Garry W
authors.initials: GW
authors.lastname: Buchko
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA. Electronic address: garavito@msu.edu.
authors.forename: R Michael
authors.initials: RM
authors.lastname: Garavito
mesh_terms: D000595:Amino Acid Sequence; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D003067:Coenzymes / Q000378:metabolism; D018360:Crystallography, X-Ray; D045302:Geobacter / Q000201:enzymology*; D006884:Hydroxybutyrate Dehydrogenase / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism
chemical_list: D003067:Coenzymes; D009249:NADP; D002237:Carbohydrate Dehydrogenases; D006884:Hydroxybutyrate Dehydrogenase; C020843:gulonate dehydrogenase; C019416:4-hydroxybutyrate dehydrogenase
keywords: Bio-inspired catalysis; GABA shunt; Protein X-ray crystallography; Structural biology; Succinic semialdehyde reductase