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. 1995 Dec 5;92(25):11666–11670. doi: 10.1073/pnas.92.25.11666

A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity.

R Chen 1, A Greer 1, A M Dean 1
PMCID: PMC40463  PMID: 8524825

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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