Structural basis for the alteration of coenzyme specificity in a malate dehydrogenase mutant.
To elucidate the structural basis for the alteration of coenzyme specificity from NADH toward NADPH in a malate dehydrogenase mutant EX7 from Thermus flavus, we determined the crystal structures at 2.0 A resolution of EX7 complexed with NADPH and NADH, respectively. In the EX7-NADPH complex, Ser42 and Ser45 form hydrogen bonds with the 2'-phosphate group of the adenine ribose of NADPH, although the adenine moiety is not seen in the electron density map. In contrast, although Ser42 and Ser45 occupy a similar position in the EX7-NADH complex structure, both the adenine and adenine ribose moieties of NADH are missing in the map. These results and kinetic analysis of site-directed mutant enzymes indicate (1) that the preference of EX7 for NADPH over NADH is ascribed to the recognition of the 2'-phosphate group by two Ser and Arg44, and (2) that the adenine moiety of NADPH is not recognized in this mutant.
Tomita
Takeo
T
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Tokyo 113-8657, Japan.
Fushinobu
Shinya
S
Kuzuyama
Tomohisa
T
Nishiyama
Makoto
M
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism; D003460:Crystallization; D006860:Hydrogen Bonding; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry*; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013824:Thermus / Q000201:enzymology
D001426:Bacterial Proteins; D003067:Coenzymes; D050505:Mutant Proteins; D009243:NAD; D012694:Serine; D009249:NADP; D008291:Malate Dehydrogenase