Alteration of coenzyme specificity of lactate dehydrogenase from Thermus thermophilus by introducing the loop region of NADP(H)-dependent malate dehydrogenase.
Previously we found that replacement of seven amino acid residues in a loop region markedly shifted the coenzyme specificity of malate dehydrogenase from NAD(H) toward NADP(H). In the present study, we replaced the seven amino acid residues in the corresponding region of an NAD(H)-dependent lactate dehydrogenase with those of NADP(H)-dependent malate dehydrogenase, and examined the coenzyme specificity of the resulting mutant enzyme. Coenzyme specificity was significantly shifted by 399-fold toward NADPH when k cat/Km(coenzyme) was used as the measure of coenzyme specificity. The effect of the replacements on coenzyme specificity is discussed based on in silico simulation of the three-dimensional structure of the lactate dehydrogenase mutant.
Tomita
Takeo
T
Biotechnology Research Center, The University of Tokyo, Tokyo, Japan.
Kuzuyama
Tomohisa
T
Nishiyama
Makoto
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D050538:Malate Dehydrogenase (NADP+) / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D007770:L-Lactate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+)