Speeding up enzyme evolution

Abstract

Enzyme evolution is thought to proceed largely by gene duplication and the propagation of slightly deleterious amino acid substitutions. Low stability and poor catalytic rates are properties shared by many newly evolved enzymes that arise as gene duplication events in which selection for stability and/or turnover is released while mutations accumulate that finally result in an alteration of function. Starting from this model, Edward Whittle and John Shanklin in the Biology Dept of Brookhaven National Laboratory (Upton, New York, USA), now show that it is possible to reenginner archetypal enzymes to achieve altered substrate specificities characteristic of recently evolved enzymes while retaining the desired stability and/or turnover characteristics of the parental paralog [J. Biol. Chem. (2001) 276, 21500–21505)]. Working with soluble castor Δ9-18:0-acyl carrier protein (ACP) desaturase, an enzyme which introduces a double bond region specifically into a saturated acyl-ACP substrate, Whittle and Shanklin used combinatorial saturation mutagenesis at six amino acid positions known to affect substrate specificity to maximize the likelihood of identifying variants with desired improvements in turnover rates. Upon identification of two key substitutions, namely T117R and G188L, researchers engineered back these changes into otherwise wild-type desaturase. Remarkably, they obtained an enzyme with significant preference for 16-carbon substrates but with kinetic parameters similar to those of wild-type desaturase for 18-carbon substrate, showing that is possible to alter the specificity of a desaturase (and most probably of other enzymes) without substantial degradation of its kinetic parameters. The engineered enzyme had a specific activity for 16-carbon 100 times higher than known natural 16-carbon specific desaturases. AR(http://www.bnl.gov/bnlweb/pubaf/pr/bnlpr061301.htm)