Characterizing KAS Enzymes Using the E. Coli Double Mutant Strain CY244

Abstract

In Escerichia coli as in plants, fatty acid biosynthesis is catalyzed by discrete enzymes of the FAS II system. The Claisen condensation of C2 units is carried out by s-ketoacyl ACP synthase (KAS) I and KAS II. These catalyze all elongation steps with the exception of the initial condensation of two C2 units catalyzed by KAS III. EcKAS I, however, is specific for elongation of C10:1 making it essential for unsaturated fatty acid biosynthesis whereas EcKAS II is specific for elongation of C16:1 to C18:1. In plants, KAS I catalyzes the elongation steps from C4 to C16 while KAS II is responsible for elongation of C16 to C18. In contrast to the bacterial FAS, double bonds are only introduced after elongation in plants. EcKAS I and II differ in their sensitivities to cerulenin, an inhibitor of condensing enzymes, EcKAS I being very sensitive while EcKAS II is intermediately sensitive; the same pattern is observed for barley KAS I and II. Fatty acid biosynthesis in plants has also been revealed to take place in mitochondria and a KAS enzyme from Arabidopsis thaliana has been cloned and localized to the mitochondria (Yasuno, R et al., in preparation). Both E. coli KAS’es have been expressed in soluble active form and their biochemical as well as basic structural characteristics have been revealed (Huang, W et al., 1998, Olsen, JG et al., 1999). With the exception of KAS II from Synechocystis sp. (Moche, M et al.,2001) all other KAS I &; II orthologs have proved recalcitrant to expression in soluble active form when overexpressed in E. coli. In this study, we have systematically explored a range of expression systems to try and overcome this problem for different KAS enzymes. By use of the E. coli temperature sensitive strain CY244 defective in fatty acid biosynthesis at 42°C we have characterized these enzymes by their ability to complement CY244 for growth at 42° in vivo and to restore elongation activity in CY244 soluble protein extracts at 42°C in vitro.