Using Ph Changes To Obtain Time-Resolved Crystallographic Structures Of Hmg-Coa Reductase

HMG-CoA reductase (HMGR) is a key enzyme in the mevalonate pathway and is involved in the biosynthesis of cholesterol. It catalyzes the conversion of HMG-CoA into Mevalonate using a double-hydride transfer using 2 equivalents of NAD(P). This pathway is also critical for the survival of antibiotic resistant pathogenic bacteria. Understanding the mechanism of bacterial HMGR can provide us with structural information that can be used to develop novel antibacterials against such bacterial infections. HMGR from pseudomonas mevalonii, with the help of large solvent channels which provide access to the active site has been shown to be active inside crystals and undergo large conformational changes that accompany the reaction in the crystal. Therefore, the HMGR in pseudomonas mevalonii is a suitable system to conduct time-resolved studies of the reaction, which can help understand its mechanism in pathogenic bacteria. We have obtained diffracting crystals of HMGR from P.Mevalonii with Mevalonate, CoA and NAD bound at the active site. However, we do not see any reaction taking place inside the crystal. The possibility is that the reaction does not proceed in the crystal environment at the crystallization buffer pH which is 6.7. A 4-fold increase in activity is seen at pH 9 in the crystallization buffer solution. Preliminary studies show the formation of HMGCoA, when the pH of the crystal is slowly changed to 9. In this study, using soaking techniques at different pH conditions between 6.7 - 9 that are suitable for providing a crystal environment where the reaction progresses slow enough to be frozen along different time-points, we aim to record x-ray diffraction data along the reaction pathway and observe the formation of the various intermediates and the structural changes that facilitate their formation in the crystal.