Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 108, Issue 46, Pages 18643-18648Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1110852108
Keywords
-
Categories
Funding
- LS9, Inc
- Ministry of Science and Technology of China [2011CBA00806]
- National Science Foundation of China [31040086]
Ask authors/readers for more resources
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 mu M, the maximum rate of free fatty acid synthesis of the FAS exceeded 100 mu M/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 mu M. The exceptions were FabI and FabZ, which increased FAS activity up to concentrations of 10 mu M; FabH and FabF, which decreased FAS activity at concentrations higher than 1 mu M; and holo-ACP and TesA, which gavemaximum FAS activity at 30 mu M 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
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available