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.