Analysis of the HMG-CoA reductase reaction mechanism by x-ray crystallography
HMG-CoA reductase (HMGR) is a critical enzyme in isoprenoid biosynthesis and a drug target for both hypercholesterolemia in humans and antibiotics targeted at gram-positive pathogens. This enzyme has an interesting reaction mechanism; it catalyzes two half-reactions to convert HMG-CoA to mevalonate using two successive hydride transfers from NADH without release of an intermediate species. HMGR from P. mevalonii is compatible with monochromatic x-ray crystallography and is functional as a 90 kDa homodimer with each monomer subdivided into a large CoA binding domain, a small NAD(H) binding domain, and a flexible C-terminal flap domain that closes over the active site. A dozen high-resolution productive and non-productive complexes are described. Results show that the thiohemiacetal intermediate is the dominant species during NAD(H) exchange and may persist to keep the enzyme and mevaldehyde intermediate primed for the second half reaction. The observation that CoA binding changes the NAD(H) binding domain similar to when NAD(H) is bound suggests that CoA binding may also function to lessen the total changes the protein experiences during NAD(H) exchange. His381 is an important catalytic residue that resides on the first helix of the C-terminal flap domain and complexes from this investigation provide structural evidence that it is the proton donor to CoA that generates free CoA-SH during the reaction. The flap domain appears to be fully ordered only when both CoA and NAD(H) are in the active site. The entrance to the flap domain can adopt one of two positions, a 'closed' position necessary for positioning the His381 in the active site, and an 'open' position that is shifted approximately 180° due to psi angle differences in Thr374 and Glu375. Intermediate structures investigated along the reaction pathway suggest that Ile377, Asp416, and a hydrogen-bonding network across the active site are important for this domain to be in an ordered conformation. In addition to generating these snapshots of the HMGR reaction by monochromatic x-ray crystallographic techniques, this enzyme is being investigated as a candidate for polychromatic time-resolved Laue crystallography. It has been determined that HMGR is compatible with the Laue technique and initial time-resolved studies have been performed.^
Cynthia V. Stauffacher, Purdue University.