PMID:8524825
| Citation |
Chen, R, Greer, A and Dean, AM (1995) A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity. Proc. Natl. Acad. Sci. U.S.A. 92:11666-70 |
|---|---|
| Abstract |
The isocitrate dehydrogenase of Escherichia coli, which lacks the Rossmann fold common to other dehydrogenases, displays a 7000-fold preference for NADP over NAD (calculated as the ratio of kcat/Km). Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to introduce six substitutions in the adenosine binding pocket that systematically shift coenzyme preference toward NAD. The engineered enzyme displays an 850-fold preference for NAD over NADP, which exceeds the 140-fold preference displayed by a homologous NAD-dependent enzyme. Of the six mutations introduced, only one is identical in all related NAD-dependent enzyme sequences--strict adherence to homology as a criterion for replacing these amino acids impairs function. Two additional mutations at remote sites improve performance further, resulting in a final mutant enzyme with kinetic characteristics and coenzyme preference comparable to naturally occurring homologous NAD-dependent enzymes. |
| Links | |
| Keywords |
Amino Acid Sequence; Binding Sites/genetics; Escherichia coli/enzymology; Escherichia coli/genetics; Isocitrate Dehydrogenase/genetics; Isocitrate Dehydrogenase/metabolism; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; NAD/metabolism; NADP/metabolism; Protein Engineering/methods; Substrate Specificity |
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