Domain swapping between Enterococcus faecalis FabN and FabZ proteins localizes the structural determinants for isomerase activity

Abstract

Anaerobic unsaturated fatty acid synthesis in bacteria occurs through the introduction of a double bond into the growing acyl chain. In the Escherichia coli model system, FabA catalyzes both the dehydration of β- hydroxydecanoyl-ACP and the isomerization of trans-2-decenoyl-ACP to cis-3-decenoyl-ACP as the essential step. A second dehydratase, FabZ, functions in acyl chain elongation but cannot carry out the isomerization reaction. Enterococcus faecalis has two highly related FabZ homologs. One of these, termed EfFabN, carries out the isomerization reaction ire vivo, whereas the other, EfFabZ, does not (Wang, H., and Cronan, J. E. (2004) J. Biol. Chem. 279, 34489-34495). We carried out a series of domain swapping and mutagenesis experiments coupled with ire vitro biochemical analyses to define the structural feature(s) that specify the catalytic properties of these two enzymes. Substitution of the β3 and β4 strands of EfFabZ with the corresponding strands from EfFabN was necessary and sufficient to convert EfFabZ into an isomerase. These data are consistent with the hypothesis that the isomerase potential of β-hydroxyacyl-ACP dehydratases is determined by the properties of the β-sheets that dictate the orientation of the central α-helix and thus the shape of the substrate binding tunnel rather than the catalytic machinery at the active site. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.