title: The biosynthetic incorporation of the intact leucine skeleton into sterol by the trypanosomatid Leishmania mexicana.
abstract: The amino acid leucine is efficiently used by the trypanosomatid Leishmania mexicana for sterol biosynthesis. The incubation of [2-(13)C]leucine with L. mexicana promastigotes in the presence of ketoconazole gave 14alpha-methylergosta-8,24(24(1))-3beta-ol as the major sterol, which was shown by mass spectrometry to contain up to six atoms of (13)C per molecule. (13)C NMR analysis of the 14alpha-methylergosta-8,24(24(1))-3beta-ol revealed that it was labeled in only six positions: C-2, C-6, C-11, C-12, C-16, and C-23. This established that the leucine skeleton is incorporated intact into the isoprenoid pathway leading to sterol; it is not converted first to acetyl-CoA, as in animals and plants, with utilization of the acetyl-CoA to regenerate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An inhibitor of HMG-CoA synthase (L-659,699) blocked the incorporation of [1-(14)C]acetate into sterol but had no inhibitory effect on [U-(14)C]leucine incorporation. The HMG-CoA reductase inhibitor lovastatin inhibited promastigote growth and [U-(14)C]leucine incorporation into sterol. The addition of unlabeled mevalonic acid (MVA) overcame the lovastatin inhibition of growth and also diluted the incorporation of [1-(14)C]leucine into sterol. These results are compatible with two routes by which the leucine skeleton may enter intact into the isoprenoid pathway. The catabolism of leucine could generate HMG-CoA that is then directly reduced to MVA for incorporation into sterol. Alternatively, a compound produced as an intermediate in leucine breakdown to HMG-CoA (e.g. dimethylcrotonyl-CoA) could be directly reduced to produce an isoprene alcohol followed by phosphorylation to enter the isoprenoid pathway post-MVA.
authors.affiliation: School of Biological Sciences, University of Liverpool, Life Sciences Building, Crown St., Liverpool L69 7ZB, United Kingdom.
authors.forename: M L
authors.initials: ML
authors.lastname: Ginger
authors.forename: M L
authors.initials: ML
authors.lastname: Chance
authors.forename: I H
authors.initials: IH
authors.lastname: Sadler
authors.forename: L J
authors.initials: LJ
authors.lastname: Goad
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D002247:Carbon Isotopes; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000037:antagonists & inhibitors; D007783:Lactones / Q000494:pharmacology; D007894:Leishmania mexicana / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D019906:Nuclear Magnetic Resonance, Biomolecular; D013261:Sterols / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D000981:Antiprotozoal Agents; D002247:Carbon Isotopes; D004791:Enzyme Inhibitors; D005231:Fatty Acids, Unsaturated; D007783:Lactones; D013261:Sterols; C054282:antibiotic 1233A; D006904:Hydroxymethylglutaryl-CoA Synthase; D007930:Leucine