Redesign of choline acetyltransferase specificity by protein engineering.
Since the development of site-directed mutagenesis techniques over 15 years ago (Zoller, M. J., and Smith, M. (1982) Nucleic Acids Res. 10, 6487-6500), it has been a goal of protein engineering to utilize the procedure to redesign existing enzyme structures to produce proteins with altered or novel catalytic properties. To date, however, the more successful achievements have relied exclusively on the availability of three-dimensional protein structure maps to direct the redesign strategies. Presently, such maps are unavailable for choline acetyltransferase and carnitine acetyltransferase, enzymes that catalyze the reversible transfer of an acetyl group from acetyl-CoA to choline and L-carnitine, respectively. A more empirical approach, based on cross-referencing substrate structure comparisons with protein alignment data, was used to redesign choline acetyltransferase to accommodate L-carnitine as an acceptor of the acetyl group. A mutant choline acetyltransferase that incorporates four amino acid substitutions from wild type, shows a substantial increase in catalytic efficiency (kcat/Km) toward L-carnitine (1,620-fold) and shifts the catalytic discrimination between choline and L-carnitine by >390,000 in favor of the latter substrate. These dramatic alterations in catalytic function demonstrate that significant success in protein redesign can be achieved in the absence of three-dimensional protein structure data.
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Molecular Biology Division, Department of Veterans Affairs Medical Center, San Francisco, California 94121, USA. ciaran@itsa.ucsf.edu
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002331:Carnitine / Q000378:metabolism; D002333:Carnitine Acyltransferases / Q000737:chemistry; D002795:Choline O-Acetyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D011994:Recombinant Proteins; D002333:Carnitine Acyltransferases; D002795:Choline O-Acetyltransferase; D002331:Carnitine