title: In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli.
abstract: Microbial fatty acid derivatives are emerging as promising alternatives to fossil fuel derived transportation fuels. Among bacterial fatty acid synthases (FAS), the Escherichia coli FAS is perhaps the most well studied, but little is known about its steady-state kinetic behavior. Here we describe the reconstitution of E. coli FAS using purified protein components and report detailed kinetic analysis of this reconstituted system. When all ketosynthases are present at 1 μM, the maximum rate of free fatty acid synthesis of the FAS exceeded 100 μM/ min. The steady-state turnover frequency was not significantly inhibited at high concentrations of any substrate or cofactor. FAS activity was saturated with respect to most individual protein components when their concentrations exceeded 1 μM. The exceptions were FabI and FabZ, which increased FAS activity up to concentrations of 10 μM; FabH and FabF, which decreased FAS activity at concentrations higher than 1 μM; and holo-ACP and TesA, which gave maximum FAS activity at 30 μM concentrations. Analysis of the S36T mutant of the ACP revealed that the unusual dependence of FAS activity on holo-ACP concentration was due, at least in part, to the acyl-phosphopantetheine moiety. MALDI-TOF mass spectrometry analysis of the reaction mixture further revealed medium and long chain fatty acyl-ACP intermediates as predominant ACP species. We speculate that one or more of such intermediates are key allosteric regulators of FAS turnover. Our findings provide a new basis for assessing the scope and limitations of using E. coli as a biocatalyst for the production of diesel-like fuels.
authors: Department of Chemical Engineering and Chemistry, Stanford University, Stanford CA 94305, USA.
authors: Xingye
authors: X
authors: Yu
authors: Tiangang
authors: T
authors: Liu
authors: Fayin
authors: F
authors: Zhu
authors: Chaitan
authors: C
authors: Khosla
mesh_terms: D000495:Allosteric Site; D002244:Carbon / Q000737:chemistry; D002384:Catalysis; D002474:Cell-Free System; D004926:Escherichia coli / Q000201:enzymology*; D064429:Fatty Acid Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D005227:Fatty Acids; D012333:RNA, Messenger; D011994:Recombinant Proteins; D002244:Carbon; D064429:Fatty Acid Synthases