Document Detail

In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli.
MedLine Citation:
PMID:  22042840     Owner:  NLM     Status:  MEDLINE    
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.
Xingye Yu; Tiangang Liu; Fayin Zhu; Chaitan Khosla
Publication Detail:
Type:  In Vitro; Journal Article; Research Support, Non-U.S. Gov't     Date:  2011-10-31
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  108     ISSN:  1091-6490     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  2011 Nov 
Date Detail:
Created Date:  2011-11-17     Completed Date:  2012-01-26     Revised Date:  2013-06-27    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  United States    
Other Details:
Languages:  eng     Pagination:  18643-8     Citation Subset:  IM    
Department of Chemical Engineering and Chemistry, Stanford University, Stanford CA 94305, USA.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Allosteric Site
Carbon / chemistry
Cell-Free System
Escherichia coli / enzymology*
Fatty Acid Synthetase Complex / chemistry*,  genetics,  metabolism*
Fatty Acids / chemistry
Plasmids / metabolism
Protein Structure, Tertiary
RNA, Messenger / metabolism
Recombinant Proteins / chemistry
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Substrate Specificity
Reg. No./Substance:
0/Fatty Acids; 0/RNA, Messenger; 0/Recombinant Proteins; 7440-44-0/Carbon; EC 6.-/Fatty Acid Synthetase Complex

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Previous Document:  Hyperpolarized 13C dehydroascorbate as an endogenous redox sensor for in vivo metabolic imaging.
Next Document:  Prototypical model for tensional wrinkling in thin sheets.