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3-hydroxy-3-methylglutaryl-CoA synthase intermediate complex observed in "real-time".
Michael J Theisen1, Ila Misra, Dana Saadat
1Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA.
Summary
3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS) uses a unique mechanism to form carbon-carbon bonds. Researchers trapped an enzyme-intermediate complex, revealing key catalytic residues and potential roles for protein dynamics in HMG-CoA biosynthesis.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Carbon-carbon bond formation via acyl-enzyme intermediates is crucial for biosynthesis pathways.
- 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS) uniquely activates a cysteine methyl group for this bond formation.
- HMGS is essential in Gram-positive bacteria and initiates human cholesterol biosynthesis.
Purpose of the Study:
- To elucidate the catalytic mechanism of HMGS by trapping an enzyme-intermediate complex.
- To identify key amino acid residues involved in substrate binding and catalysis.
- To understand the role of protein dynamics in the physiological reaction.
Main Methods:
- Enzyme crystallography of Staphylococcus aureus HMGS.
- Cryo-cooling of enzyme crystals at different time points during the back-reaction.
- Analysis of enzyme-intermediate complexes with bound acetoacetyl-CoA.
Main Results:
- A complex of acetylated HMGS and acetoacetyl-CoA was successfully trapped.
- Structural data revealed that active-site glutamic acid (Glu-79) functions as a general base.
- Glu-79 is involved in both the condensation reaction and the hydrolytic release of HMG-CoA.
Conclusions:
- The trapped complex provides insights into the mechanism of the physiological HMGS reaction.
- Specific residues, like Glu-79, play critical roles in catalysis and substrate binding.
- Protein dynamics and inter-protomer interactions may be important for HMGS function.