A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity.
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.
Department of Biological Chemistry, Chicago Medical School, North Chicago, IL 60064, USA.
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Chen
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Greer
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Dean
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007521:Isocitrate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity
D009243:NAD; D009249:NADP; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)