Isolation and characterization of beta-ketoacyl-acyl carrier protein reductase (fabG) mutants of Escherichia coli and Salmonella enterica serovar Typhimurium.
FabG, beta-ketoacyl-acyl carrier protein (ACP) reductase, performs the NADPH-dependent reduction of beta-ketoacyl-ACP substrates to beta-hydroxyacyl-ACP products, the first reductive step in the elongation cycle of fatty acid biosynthesis. We report the first documented fabG mutants and their characterization. By chemical mutagenesis followed by a tritium suicide procedure, we obtained three conditionally lethal temperature-sensitive fabG mutants. The Escherichia coli [fabG (Ts)] mutant contains two point mutations: A154T and E233K. The beta-ketoacyl-ACP reductase activity of this mutant was extremely thermolabile, and the rate of fatty acid synthesis measured in vivo was inhibited upon shift to the nonpermissive temperature. Moreover, synthesis of the acyl-ACP intermediates of the pathway was inhibited upon shift of mutant cultures to the nonpermissive temperature, indicating blockage of the synthetic cycle. Similar results were observed for in vitro fatty acid synthesis. Complementation analysis revealed that only the E233K mutation was required to give the temperature-sensitive growth phenotype. In the two Salmonella enterica serovar Typhimurium fabG(Ts) mutants one strain had a single point mutation, S224F, whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the twofold axes of symmetry at the dimer interfaces in this homotetrameric protein, suggesting that the quaternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature.
Lai
Chiou-Yan
CY
Department of Microbiology, University of Illinois, Urbana, Illinois 61801, USA.
Cronan
John E
JE
D000213:Acyl Carrier Protein / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005227:Fatty Acids / Q000096:biosynthesis; D005816:Genetic Complementation Test; D008958:Models, Molecular; D009154:Mutation; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013696:Temperature
D000213:Acyl Carrier Protein; D005227:Fatty Acids; D000429:Alcohol Oxidoreductases; C023744:acetoacetyl-CoA reductase