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.
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0795, Japan.
Junji
J
Hayashi
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0795, Japan.
Kaori
K
Yamamoto
Department of Bioscience, School of Agriculture, Tokai University, Aso, Kumamoto, 869-1404, Japan.
Kazunari
K
Yoneda
Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology, 5-16-1 Ohmiya, Asahi-ku, Osaka, 535-8585, Japan.
Toshihisa
T
Ohshima
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0795, Japan.
Haruhiko
H
Sakuraba
D020816:Amino Acid Motifs; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005993:Glycerolphosphate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D041543:Pyrobaculum / Q000737:chemistry* / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D019843:Archaeal Proteins; D003067:Coenzymes; D021122:Protein Subunits; D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D005993:Glycerolphosphate Dehydrogenase; C098215:glycerol-1-phosphate dehydrogenase
NADPH; Pyrobaculum calidifontis; archaea; coenzyme preference; hyperthermophile; sn-glycerol-1-phosphate dehydrogenase