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Originally published In Press as doi:10.1074/jbc.M102129200 on April 9, 2001

J. Biol. Chem., Vol. 276, Issue 24, 21500-21505, June 15, 2001
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Engineering Delta 9-16:0-Acyl Carrier Protein (ACP) Desaturase Specificity Based on Combinatorial Saturation Mutagenesis and Logical Redesign of the Castor Delta 9-18:0-ACP Desaturase*

Edward Whittle and John ShanklinDagger

From the Biology Department, Brookhaven National Laboratory, Upton, New York 11973

Six amino acid locations in the soluble castor Delta 9-18:0-acyl carrier protein (ACP) desaturase were identified that can affect substrate specificity. Combinatorial saturation mutagenesis of these six amino acids, in conjunction with selection, using an unsaturated fatty acid auxotroph system, led to the isolation of variants with up to 15-fold increased specific activity toward 16-carbon substrates. The most improved mutant, com2, contained two substitutions (T117R/G188L) common to five of the 19 complementing variants subjected to further analysis. These changes, when engineered into otherwise wild-type 18:0-ACP desaturase to make mutant 5.2, produced a 35-fold increase in specific activity with respect to 16-carbon substrates. Kinetic analysis revealed changes in both kcat and Km that result in an 82-fold improvement in specificity factor for 16-carbon substrate compared with wild-type enzyme. Improved substrate orientation apparently compensated for loss of binding energy that results from the loss of desolvation energy for 16-carbon substrates. Mutant 5.2 had specific activity for 16-carbon substrates 2 orders of magnitude higher than those of known natural 16-carbon specific desaturases. These data support the hypothesis that it should be possible to reengineer archetypal enzymes to achieve substrate specificities characteristic of recently evolved enzymes while retaining the desired stability and/or turnover characteristics of a parental paralog.

* This work was supported by the Office of Basic Energy Sciences of the United States Department of Energy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Biology, Bldg. 463, Brookhaven National Laboratory, 50 Bell Ave., Upton, NY 11973. Tel.: 631-344-3414; Fax: 631-344-3407; E-mail: shanklin@bnl.gov.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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