title: An Eight-Residue Deletion in Escherichia coli FabG Causes Temperature-Sensitive Growth and Lipid Synthesis Plus Resistance to the Calmodulin Inhibitor Trifluoperazine.
abstract: FabG performs the NADPH-dependent reduction of β-keto acyl-acyl carrier protein substrates in the elongation cycle of fatty acid synthesis. We report the characterization of a temperature-sensitive mutation (fabGΔ8) inEscherichia colifabGthat results from an in-frame 8-amino-acid residue deletion in the α6/α7 subdomain. This region forms part of one of the two dimerization interfaces of this tetrameric enzyme and is reported to undergo significant conformational changes upon cofactor binding, which define the entrance to the active-site cleft. The activity of the mutant enzyme is extremely thermolabile and is deficient in forming homodimers at nonpermissive temperatures with a corresponding decrease in fatty acid synthesis bothin vivoandin vitroSurprisingly, thefabGΔ8strain reverts to temperature resistance at a rate reminiscent of that of a point mutant with intragenic pseudorevertants located either on the 2-fold axes of symmetry or at the mouth of the active-site cleft. ThefabGΔ8mutation also confers resistance to the calmodulin inhibitor trifluoperazine and renders the enzyme extremely sensitive to Ca2+in vitroWe also observed a significant alteration in the lipid A fatty acid composition offabGΔ8strains but only in anlpxCbackground, probably due to alterations in the permeability of the outer membrane. These observations provide insights into the structural dynamics of FabG and hint at yet another point of regulation between fatty acid and lipid A biosynthesis.IMPORTANCEMembrane lipid homeostasis and its plasticity in a variety of environments are essential for bacterial survival. Since lipid biosynthesis in bacteria and plants is fundamentally distinct from that in animals, it is an ideal target for the development of antibacterial therapeutics. FabG, the subject of this study, catalyzes the first cofactor-dependent reduction in this pathway and is active only as a tetramer. This study examines the interactions responsible for tetramerization through the biochemical characterization of a novel temperature-sensitive mutation caused by a short deletion in an important helix-turn-helix motif. The mutant strain has altered phospholipid and lipid A compositions and is resistant to trifluoperazine, an inhibitor of mammalian calmodulin. Understanding its structural dynamics and its influence on lipid A synthesis also allows us to explore lipid homeostasis as a mechanism for antibiotic resistance.
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
authors.forename: Swaminath
authors.initials: S
authors.lastname: Srinivas
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA j-cronan@life.illinois.edu.|Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
authors.forename: John E
authors.initials: JE
authors.lastname: Cronan
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002118:Calcium / Q000633:toxicity; D024881:Drug Resistance, Bacterial / Q000528:radiation effects*; D004795:Enzyme Stability / Q000528:radiation effects; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development* / Q000528:radiation effects*; D050356:Lipid Metabolism / Q000528:radiation effects*; D055503:Protein Multimerization; D017384:Sequence Deletion; D013489:Suppression, Genetic; D013696:Temperature; D014268:Trifluoperazine / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D014268:Trifluoperazine; D000429:Alcohol Oxidoreductases; C023744:acetoacetyl-CoA reductase; D002118:Calcium
keywords: antibiotic resistance; calcium; deletion mutant; dimer interface; fatty acid synthesis; lipid A; temperature sensitive