Structural Constraints in Protein Engineering

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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, 11 666–11670]. 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 Å 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 rational design of active‐site residues based on X‐ray structures and random substitutions at surrounding residues provides an efficient means to improve enzyme preference and catalytic efficiency towards novel substrates.