RRC ID 48524
Author Hayashi J, Yamamoto K, Yoneda K, Ohshima T, Sakuraba H.
Title Unique coenzyme binding mode of hyperthermophilic archaeal sn-glycerol-1-phosphate dehydrogenase from Pyrobaculum calidifontis.
Journal Proteins
Abstract A gene encoding an sn-glycerol-1-phosphate dehydrogenase (G1PDH) was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. In contrast to conventional G1PDHs, the expressed enzyme showed strong preference for NADH: the reaction rate (Vmax ) with NADPH was only 2.4% of that with NADH. The crystal structure of the enzyme was determined at a resolution of 2.45 Å. The asymmetric unit consisted of one homohexamer. Refinement of the structure and HPLC analysis showed the presence of the bound cofactor NADPH in subunits D, E, and F, even though it was not added in the crystallization procedure. The phosphate group at C2' of the adenine ribose of NADPH is tightly held through the five biased hydrogen bonds with Ser40 and Thr42. In comparison with the known G1PDH structure, the NADPH molecule was observed to be pushed away from the normal coenzyme binding site. Interestingly, the S40A/T42A double mutant enzyme acquired much higher reactivity than the wild-type enzyme with NADPH, which suggests that the biased interactions around the C2'-phosphate group make NADPH binding insufficient for catalysis. Our results provide a unique structural basis for coenzyme preference in NAD(P)-dependent dehydrogenases. Proteins 2016; 84:1786-1796. © 2016 Wiley Periodicals, Inc.
Volume 84(12)
Pages 1786-1796
Published 2016-12-1
DOI 10.1002/prot.25161
PMID 27616573
MeSH Amino Acid Motifs Archaeal Proteins / chemistry* Archaeal Proteins / genetics Archaeal Proteins / metabolism Binding Sites Cloning, Molecular Coenzymes / chemistry* Coenzymes / metabolism Crystallography, X-Ray Escherichia coli / genetics Escherichia coli / metabolism Gene Expression Glycerolphosphate Dehydrogenase / chemistry* Glycerolphosphate Dehydrogenase / genetics Glycerolphosphate Dehydrogenase / metabolism Hydrogen Bonding Kinetics Models, Molecular NAD / chemistry* NAD / metabolism NADP / chemistry* NADP / metabolism Protein Binding Protein Interaction Domains and Motifs Protein Multimerization Protein Structure, Secondary Protein Subunits / chemistry* Protein Subunits / genetics Protein Subunits / metabolism Pyrobaculum / chemistry* Pyrobaculum / enzymology Recombinant Proteins / chemistry Recombinant Proteins / genetics Recombinant Proteins / metabolism Thermodynamics
IF 2.828
Times Cited 0
General Microbes JCM 11548