Unique coenzyme binding mode of hyperthermophilic archaeal sn-glycerol-1-phosphate dehydrogenase from Pyrobaculum calidifontis.
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.
['Hayashi|Junji|J|', 'Yamamoto|Kaori|K|', 'Yoneda|Kazunari|K|', 'Ohshima|Toshihisa|T|', 'Sakuraba|Haruhiko|H|']
D020816:Amino Acid Motifs D019843:Archaeal Proteins D001665:Binding Sites D003001:Cloning, Molecular D003067:Coenzymes D018360:Crystallography, X-Ray D004926:Escherichia coli D015870:Gene Expression D005993:Glycerolphosphate Dehydrogenase D006860:Hydrogen Bonding D007700:Kinetics D008958:Models, Molecular D009243:NAD D009249:NADP D011485:Protein Binding D054730:Protein Interaction Domains and Motifs D055503:Protein Multimerization D017433:Protein Structure, Secondary D021122:Protein Subunits D041543:Pyrobaculum D011994:Recombinant Proteins D013816:Thermodynamics
hyperthermophile sn-glycerol-1-phosphate dehydrogenase Pyrobaculum calidifontis NADPH coenzyme preference archaea