Award Abstract #0133826
PECASE: Mechanistic Studies on Cyclopropane Fatty Acid Synthase

NSF Org:	MCB Division of Molecular and Cellular Biosciences
Initial Amendment Date:	January 31, 2002
Latest Amendment Date:	June 6, 2007
Award Number:	0133826
Award Instrument:	Continuing grant
Program Manager:	David A. Rockcliffe MCB Division of Molecular and Cellular Biosciences BIO Directorate for Biological Sciences
Start Date:	March 1, 2002
Expires:	February 29, 2008 (Estimated)
Awarded Amount to Date:	$557295
Investigator(s):	Squire Booker sjb14@psu.edu (Principal Investigator)
Sponsor:	Pennsylvania State Univ University Park 110 Technology Center Building UNIVERSITY PARK, PA 16802 814/865-1372
NSF Program(s):	MOLECULAR BIOCHEMISTRY, BIOMOLECULAR SYSTEMS, INSTRUMENTAT & INSTRUMENT DEVP
Field Application(s):
Program Reference Code(s):	SMET, BIOT, 9251, 9183, 9178, 9138, 1228, 1187, 1166, 1164, 1076, 1045
Program Element Code(s):	1166, 1144, 1108

ABSTRACT



Proposal Title: PECASE: Mechanistic Studies on Cyclopropane Fatty Acid Synthase

Institution: Pennsylvania State Univ University Park

S-adenosyl-L-methionine (AdoMet) is one of the most versatile cellular metabolites used in enzymatic catalysis. Until relatively recently, it has been appreciated primarily as a cellular methylating agent, since it is the primary source of methyl groups for a broad spectrum of biological compounds, including DNA, RNA, proteins, lipids, carbohydrates, and a diverse array of small molecules. AdoMet is also involved in a variety of other interesting transformations. In one class of enzymes, it functions in concert with iron-sulfur clusters to generate enzyme-bound radicals that are intermediates in certain enzymatic reactions. In the project described herein, it functions as a donor of a methylene group rather than a methyl group, in a fascinating reaction catalyzed by the enzyme cyclopropane fatty acid synthase from Escherichia coli. The second substrate is an isolated and unactivated cis olefin present in the unsaturated fatty acid acyl chains of phospholipids. In contrast to methyl transfer, there is very little precedent in the biochemical literature for methylene transfer from AdoMet. All polar mechanisms for the reaction would be expected to involve intermediates that are very high in energy. In addition, since most cellular phospholipids are constituents of phospholipid bilayers, wherein their fatty acid chains are sequestered from the aqueous milieu, it is unclear how this enzyme catalyzes a reaction between two substrates of opposing solubilities. The goal of this project is to address these intriguing questions using a variety of kinetic, mechanistic, and physical techniques, which if successful, will contribute significantly to the general understanding of enzyme reaction mechanisms.

This project was originally funded as a CAREER award, and was converted to a Presidential Early Career Award for Engineers and Scientists (PECASE) award in May 2004.

PUBLICATIONS PRODUCED AS A RESULT OF THIS RESEARCH

Iwig, D.F., Booker, S.J.. "Insight into the polar reactivity of the onium chalcogen analogs of S-adenosyl-L- methionine," Biochemistry, v.43, 2004, p. 13496.

Iwig, D.F., Grippe, A.T., McIntryre, T.A., Booker, S. J.. "Isotope and elemental effects indicate a rate-limiting methyl transfer as the initial step in the reaction catalyzed by Escherichia coli cyclopropane fatty acid synthase," Biochemistry, v.43, 2004, p. 13510.

Iwig, DF., Uchida, A., Stromberg, JA., Booker, SJ.. "The activity of Escherichia coli cyclopropane fatty acid synthase depends on the presence of bicarbonate," Journal of the American Chemical Society, v.127, 2005, p. 11612.

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