Domain swapping between Enterococcus faecalis FabN and FabZ proteins localizes the structural determinants for isomerase activity.
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 beta-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 in 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 in vitro biochemical analyses to define the structural feature(s) that specify the catalytic properties of these two enzymes. Substitution of the beta3 and beta4 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 beta-hydroxyacyl-ACP dehydratases is determined by the properties of the beta-sheets that dictate the orientation of the central alpha-helix and thus the shape of the substrate binding tunnel rather than the catalytic machinery at the active site.
Department of Infectious Diseases, St. Jude Children's Hospital, Memphis, Tennessee 38105-2794, USA.
Ying-Jie
YJ
Lu
Stephen W
SW
White
Charles O
CO
Rock
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D013293:Enterococcus faecalis / Q000201:enzymology*; D007535:Isomerases / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D019745:cis-trans-Isomerases / Q000737:chemistry* / Q000502:physiology*
D001426:Bacterial Proteins; D017931:DNA Primers; C490371:FabZ protein, Enterococcus faecalis; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D007535:Isomerases; D019745:cis-trans-Isomerases