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Structural studies of substrate binding and catalysis in Pseudomonas mevalonii HMG-CoA reductase

Purdue University
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  • Biophysics
  • General
  • Biology


3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) catalyzes the first committed step in the mevalonate pathway for isoprenoid biosynthesis in eukaryotes, a pathway whose end products include cholesterol. This enzyme is widely distributed throughout nature, with sequences falling into two classes of enzymes corresponding roughly to eukaryotic forms and prokaryotic forms, with archaebacterial forms falling into both classes. The enzyme is complex in structure, with multiple oligomeric forms depending on the class of the enzyme, but possessing a minimal functional unit of a dimer. HMGR from the soil bacterium Pseudomonas mevalonii also has a flexible C-terminal flap domain that only folds upon binding of substrate and NAD(H) cofactor. It is similarly complex in function, catalyzing the stereospecific four-electron reduction of (S)-HMG-CoA to (R)-mevalonate, with the accompanying turnover of two equivalents of NADH, without release of an intermediate. Despite crystal structures of P. mevalonii HMGR at 2.8Å with and without substrates and cofactors bound, substrate binding determinants and requirements for C-terminal flap closure remained uncertain. ^ Crystal structures of the native enzyme at 2.2Å resolution, a binary complex with (S)-HMG-CoA bound at 2.1Å resolution, and a productive ternary complex of mevalonate and NAD+ bound at 2.7Å resolution have now been solved, providing a detailed view of substrate binding. The productive complex with mevalonate and NAD+ bound retains a partially bound cofactor molecule, raising the question of whether it is truly a catalytically competent complex. Steady-state fluorescence spectroscopy experiments confirm results from the crystal structures, suggesting that a complex able to catalyze the full enzymatic reaction is required for full C-terminal flap closure, and that the reaction does not proceed unless all components required for the full reaction are present. ^

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