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 isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass 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 cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control.
Montalvetti
A
A
Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, C/Ventanilla 11, 18001-Granada, Spain.
Peña-Díaz
J
J
Hurtado
R
R
Ruiz-Pérez
L M
LM
González-Pacanowska
D
D
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002845:Chromatography; D002850:Chromatography, Gel; D002874:Chromosome Mapping; D017886:Durapatite / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology*; D008148:Lovastatin / Q000494:pharmacology; D008798:Mevalonic Acid / Q000494:pharmacology; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012323:RNA Processing, Post-Transcriptional; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D017886:Durapatite; D008148:Lovastatin; D006903:Hydroxymethylglutaryl CoA Reductases; D007654:Ketoconazole; D008798:Mevalonic Acid; D000645:Ammonium Sulfate