Squire J. Booker Associate Professor of Biochemistry and Molecular Biology 330 South Frear Lab University Park, PA 16802 Telephone: (814) 865-8793 E-mail:sjb14@psu.edu B.A. in chemistry, Austin College, 1987 Ph.D. in biochemistry, Massachusetts Institute of Technology, 1994 Booker Lab Web Site

Mechanisms of Cofactor Action in Enzymatic Reactions and Chemical Biology

Enzymes carry out biochemical reactions with astronomical rate enhancements and amazing stereoselectivities, mediating the huge quantity and variety of cellular transformations that constitute what is vaguely termed "life." Our laboratory is endeavoring to understand at the detailed molecular level the reaction mechanisms that are employed by various enzymes. In particular, we are interested in the manner in which enzymes bind and use cofactors-whether simple metal ions, complex metal clusters, or small molecules-to increase their catalytic capabilities beyond that which is supported by the functional groups of the twenty naturally occurring amino acids. To address these issues, we use a traditional enzymological approach in combination with various spectroscopies, small-scale organic synthesis, and fast reaction kinetics.

Presently, we are focusing on enzymes that use S-adenosyl-L-methionine (AdoMet) as a substrate or cofactor in reactions that are "nonclassical" for this metabolite. AdoMet is the primary source of methyl groups for a broad spectrum of biological compounds, including DNA, RNA, proteins, lipids, carbohydrates, and many small molecules. The enzymatic mechanism of AdoMet-dependent transmethylation reactions is well documented and involves attack of a nucleophile on the methyl moiety of AdoMet with concomitant release of the thioether S-adenosyl-L-homocysteine (AdoHcy). Nature has cleverly devised a way to exploit the unique properties of the sulfonium of AdoMet for other purposes, which we call nonclassical AdoMet-dependent enzymatic reactions. Representative enzymes from two different nonclassical AdoMet-dependent reactions are currently under study. These include lysine 2,3-aminomutase, lipoic acid synthase, and cyclopropane fatty acid synthase.

AdoMet-dependent Methylene Transfer

Cyclopropane Fatty Acid Synthase. CFA synthase catalyzes the cyclopropanation of cis double bonds in acyl chains of phospholipid bilayer unsaturated fatty acids. The methylene carbon of the cyclopropane ring is derived from AdoMet. The biosynthesis of these phospholipid cyclopropane rings occurs as E. coli enter stationary phase; however, the precise role for the modification is unknown. This modification is present in many different bacteria, including the mycolic acid portion of the cell wall of Mycobacterium tuberculosis. Although the mechanism of enzymatic methyl transfer is straightforward, enzymatic methylene transfer to form a cyclopropane ring is mechanistically challenging, and suggests novel chemistry, or "interesting" high-energy intermediates. We are currently endeavoring to ascertain whether methyl transfer to the phospholipid is the initial chemical step, or whether intermediates containing modified forms of AdoMet are operative.

AdoMet-dependent Radical Generation Relatively recently, a new role for AdoMet has emerged. It is used by a group of metalloenzymes to generate various carbon-centered radicals, which are obligatory enzyme-bound intermediates in their respective reactions. The oxidizing species is thought to be a high-energy 5'-deoxyadenosyl radical that is generated via a reductive cleavage of AdoMet. All enzymes within this class contain Fe₄S₄ clusters, which are believed to function intimately in the radical-generating process. We are presently studying two enzymes within this class. Lipoic acid synthase catalyzes the insertion of two sulfur atoms into octanoic acid to give lipoic acid, which is an essential component of several multienzyme complexes that are involved in the oxidative decarboxylation of various a-keto acids and glycine. Lysine 2,3-aminomutase catalyzes the interconversion of L-a-lysine and L-b-lysine, a process that allows the bacterium from which it is isolated to use the amino acid as its sole source of carbon and nitrogen. b-lysine, as well as several other b-amino acids, which can be formed by a similar mechanism, are components of several important antibiotics.

Representative Publications:

• Cicchillo, R. M., and Booker, S. J. (2005) Mechanistic investigations of lipoic acid biosynthesis in Escherichia coli: both sulfur atoms in lipoic acid are contributed by the same lipoyl synthase polypeptide. J. Am. Chem. Soc. (accepted).
• Nesbitt, N. M., Baleanu-Gogonea, C., Cicchillo, R. M., Goodson, K., Iwig, D. F., Broadwater, J. A., Haas, J. A., Fox, B. G., and Booker, S. J. (2004) Expression, purification, and physical characterization of Escherichia coli lipoyl(octanoyl)transferase. Protein Exp. Purif. 39, 269-282.
• Iwig, D. F., Grippe, A. T., McIntyre, T. A., and Booker, S. J. (2004) 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 43, 13510-13524.
• Iwig, D. F., and Booker, S. J. (2004) Insight into the polar reactivity of the onium chalcogen analogs of S-adenosyl-L-methionine.  Biochemistry 43, 13496-13509..
• Cicchillo, R. M., Lee, K.-H., Baleanu-Gogonea, C., Nesbitt, N. M., Krebs, C., and Booker, S. J. (2004) Escherichia coli lipoyl synthase binds two distinct [4Fe–4S] clusters per polypeptide.  Biochemistry 43, 11770-11781.
• Cicchillo, R. M., Baker, M. A., Schnitzer, E. J., Newman, E. B., Krebs, C., and Booker, S. J. (2004) Escherichia coli L-serine deaminase requires a [4Fe–4S] cluster in catalysis.  J. Biol. Chem. 279, 32418-32425.
• Cicchillo, R. M., Iwig, D. F., Jones, A. D., Nesbitt, N. M., Baleanu-Gogonea, C., Souder, M. G., Tu, L., and Booker, S. J. (2004) Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid. Biochemistry 43, 6378-6386.
• Booker, S. J. (2004) Enzymatic free radical reactions. Nature Encyclopedia of Life Sciences. www.ELS.net
• Booker, S. J. (2004) Unraveling the pathway of lipoyl biosynthesis.  Chem. Biol. 11, 10-12.
• Frey, P. A., and Booker, S. J.  (2001) Radical mechanisms of S-adenosylmethionine-dependent enzymes.   Adv Protein Chem. 58, 1-45.
• Cosper, N. J., Booker, S. J.,, Ruzicka, F. J., Frey, P. A., and Scott, R. A.  (2000) Direct FeS cluster involvement in generation of a radical in lysine 2,3-aminomutase.  Biochemistry 39, 15668-15673.
• Wu, W., Booker, S., Lieder, K. W., Bandarian, V., Reed, G. H., and Frey, P. A.  (2000) Lysine 2,3-aminomutase and (E)--4,5-didehydrolysine:  Characterization of an allylic analog of a substrate-based radical in the catalytic mechanism.  Biochemistry, 39, 9561-9570.
• Frey, P. A., and Booker, S. J. (1999)  Radical intermediates in the reaction of lysine 2,3-aminomutase.  Advances in Free Radical Chemistry.  Vol 2, pp. 1-43.  JAI Press, Inc. Greenwich, CT.

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