3-hydroxy-3-methylglutaryl–CoA synthase intermediate complex observed in “real-time”
- Michael J. Theisen*,
- Ila Misra†,
- Dana Saadat†,‡,
- Nino Campobasso§,
- Henry M. Miziorko†,¶,∥, and
- David H. T. Harrison*,**
- *Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064; †Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226; and §Computational, Analytical, and Structural Sciences, GlaxoSmithKline, King of Prussia, PA 19406
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Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved September 27, 2004 (received for review August 8, 2004)
Abstract
The formation of carbon–carbon bonds via an acyl-enzyme intermediate plays a central role in fatty acid, polyketide, and isoprenoid biosynthesis. Uniquely among condensing enzymes, 3-hydroxy-3-methylglutaryl (HMG)–CoA synthase (HMGS) catalyzes the formation of a carbon–carbon bond by activating the methyl group of an acetylated cysteine. This reaction is essential in Gram-positive bacteria, and represents the first committed step in human cholesterol biosynthesis. Reaction kinetics, isotope exchange, and mass spectroscopy suggest surprisingly that HMGS is able to catalyze the “backwards” reaction in solution, where HMG–CoA is cleaved to form acetoacetyl-CoA (AcAc–CoA) and acetate. Here, we trap a complex of acetylated HMGS from Staphylococcus aureus and bound acetoacetyl-CoA by cryo-cooling enzyme crystals at three different times during the course of its back-reaction with its physiological product (HMG–CoA). This nonphysiological “backwards” reaction is used to understand the details of the physiological reaction with regards to individual residues involved in catalysis and substrate/product binding. The structures suggest that an active-site glutamic acid (Glu-79) acts as a general base both in the condensation between acetoacetyl-CoA and the acetylated enzyme, and the hydrolytic release of HMG–CoA from the enzyme. The ability to trap this enzyme–intermediate complex may suggest a role for protein dynamics and the interplay between protomers during the normal course of catalysis.
Footnotes
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↵ ∥ To whom correspondence regarding the isotope exchange data may be addressed. E-mail: miziorkoh{at}umkc.edu. **To whom correspondence may be addressed. E-mail: david.harrison{at}rosalindfranklin.edu.
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↵ ‡ Present address: Department of Internal Medicine, University of California, Irvine, CA 90815.
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↵ ¶ Present address: Division of Molecular Biology and Biochemistry, University of Missouri, Kansas City, MO 64110.
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Author contributions: H.M.M. and D.H.T.H. designed research; M.J.T., I.M., D.S., and D.H.T.H. performed research; N.C. contributed new reagents/analytic tools; M.J.T., I.M., H.M.M., and D.H.T.H. analyzed data; and M.J.T. and D.H.T.H. wrote the paper.
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This paper was submitted directly (Track II) to the PNAS office.
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Abbreviations: HMG, 3-hydroxy-3-methylglutaryl; HMGS, HMG–CoA synthase; AcAc–CoA, acetoacetyl–CoA; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight.
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Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 1XPk, 1XPL, and 1XPM).
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See Commentary on page 16399.
- Copyright © 2004, The National Academy of Sciences
