A modified consensus approach to mutagenesis inverts the cofactor specificity of Bacillus stearothermophilus lactate dehydrogenase.
Lactate dehydrogenase from Bacillus stearothermophilus is specific for NAD+. There have been several attempts to alter the cofactor specificity of this enzyme, but these have yielded enzymes with relatively low activities that still largely prefer NAD+. A modified consensus approach was used to create a library of phylogenetically preferred amino acids situated near the cofactor binding site, and variants were screened for their ability to utilize NMN+. A triple mutant (Mut31) was discovered that proved to be more catalytically efficient than wild-type. Mut31 was also better at utilizing NAD+ than the wild-type enzyme and was weakly active with NADP+ and NMN+. An analysis of single amino acid substitutions suggested that all three mutations worked in a concerted fashion to yield robust cofactor utilization. When two previously identified amino acid substitutions were introduced into the Mut31 background, the resultant quintuply substituted enzyme not only utilized NADP+ far better than the wild-type enzyme, it actually inverted its preference for NAD+ and NADP+.
Flores
Humberto
H
Instituto de Biotecnología/UNAM, Apartado Postal 510-3, Cuernavaca, Morelos, 62271, México.
Ellington
Andrew D
AD
D019943:Amino Acid Substitution; D016384:Consensus Sequence; D019020:Directed Molecular Evolution; D005635:Fructosediphosphates / Q000378:metabolism; D015723:Gene Library; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010802:Phylogeny; D013379:Substrate Specificity
D005635:Fructosediphosphates; D009243:NAD; D009249:NADP; D007770:L-Lactate Dehydrogenase