Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase

In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isopr enoid compounds. Here we report the isolation and overexpression of the gen e coding for HMG-CoA reductase from Leishmania major. The protein from Leis hmania lacks the membrane domain characteristic of eukaryotic cells but exh ibits sequence similarity with eukaryotic reductases. Highly purified prote in was achieved by ammonium sulphate precipitation followed by chromatograp hy on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K-m values for the overall reaction of 40.3+/-5.8 mu M for (R,S)-HMG-CoA and 81.4+/-5.3 mu M for NADPH; V-max was 33.55+/-1.8 uni ts . mg(-1). Gel-filtration experiments suggested an apparent molecular mas s of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote ce lls was studied. Neither mevalonic acid nor serum sterols appear to modulat e enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot a nalyses indicate that this activation is apparently performed via posttrans criptional control.