PMID: 9428712Jan 15, 1998Paper

Structural constraints in protein engineering--the coenzyme specificity of Escherichia coli isocitrate dehydrogenase

European Journal of Biochemistry
R ChenA M Dean

Abstract

In a previous study we reported on the successful inversion of coenzyme specificity in isocitrate dehydrogenase (IDH) from NADP to NAD [Chen, R., Greer, A. & Dean, A. M. (1995) A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity, Proc. Natl Acad. Sci. USA 92, 11666-11670]. Here, we explore alternative means to generate NAD dependence in the NADP-dependent scaffold of Escherichia coli IDH. The results reveal that engineering a preference for NAD is constrained by the architecture of the IDH coenzyme binding pocket and confirms that the substituted Asp344 in the engineered enzyme is the major determinant of coenzyme specificity. Mutations in the 316-325 loop, which forms part of the coenzyme binding site, reduce activity through transmission of long-range conformational changes into the active site some 14 A distant. Conformational changes seen upon substituting Cys332-->Tyr are not directly involved with improving activity. Replacements at Cys201 reveal that subtle changes in the packing of hydrophobic residues (Met and Ile versus Leu) can elicit markedly different responses. We caution against using sequence alignments as the sole guide for mutagenesis and show how a combination of ration...Continue Reading

References

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Citations

Nov 15, 2005·The Protein Journal·Adoración Rodríguez-ArnedoMaría-José Bonete
Jan 9, 2001·Trends in Biotechnology·R Chen
Sep 21, 2011·Acta Crystallographica. Section D, Biological Crystallography·Navdeep S SidhuGeorge M Sheldrick
Feb 24, 2001·Protein Science : a Publication of the Protein Society·R Chen, S S Jeong
Jul 22, 2006·Molecular Biology and Evolution·Arlin Stoltzfus
Oct 17, 2009·Bioorganic & Medicinal Chemistry·Eriko NangoTadashi Eguchi

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