The Biosynthetic Incorporation of the Intact Leucine Skeleton into Sterol by the Trypanosomatid Leishmania mexicana

Michael L. Ginger, Michael L. Chance, Ian H. Sadler, L. John Goad

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

The amino acid leucine is efficiently used by the trypanosomatid Leishmania mexicana for sterol biosynthesis. The incubation of [2- 13C]leucine with L. mexicana promastigotes in the presence of ketoconazole gave 14α-methylergosta-8,24(241)-3β-ol as the major sterol, which was shown by mass spectrometry to contain up to six atoms of 13C per molecule. 13C NMR analysis of the 14α-methylergosta8,24(241)-3β-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 [1-14C]acetate into sterol but had no inhibitory effect on [U-14C]leucine incorporation. The HMG-CoA reductase inhibitor lovastatin inhibited promastigote growth and [U-14C]leucine incorporation into sterol. The addition of unlabeled mevalonic acid (MVA) overcame the lovastatin inhibition of growth and also diluted the incorporation of [1-14C]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 (eg. dimethylcrotonyl-CoA) could be directly reduced to produce an isoprene alcohol followed by phosphorylation to enter the isoprenoid pathway post-MVA.

Original languageEnglish
Pages (from-to)11674-11682
Number of pages9
JournalJournal of Biological Chemistry
Volume276
Issue number15
DOIs
Publication statusPublished - 13 Apr 2001
Externally publishedYes

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Leishmania mexicana
Sterols
Skeleton
Leucine
Coenzyme A
Mevalonic Acid
Terpenes
Lovastatin
Acetyl Coenzyme A
antibiotic 1233A
Phosphorylation
Ketoconazole
Biosynthesis
Growth
Mass spectrometry
Mass Spectrometry
Oxidoreductases
Animals
Acetates
Alcohols

Cite this

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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- 13C]leucine with L. mexicana promastigotes in the presence of ketoconazole gave 14α-methylergosta-8,24(241)-3β-ol as the major sterol, which was shown by mass spectrometry to contain up to six atoms of 13C per molecule. 13C NMR analysis of the 14α-methylergosta8,24(241)-3β-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 [1-14C]acetate into sterol but had no inhibitory effect on [U-14C]leucine incorporation. The HMG-CoA reductase inhibitor lovastatin inhibited promastigote growth and [U-14C]leucine incorporation into sterol. The addition of unlabeled mevalonic acid (MVA) overcame the lovastatin inhibition of growth and also diluted the incorporation of [1-14C]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 (eg. dimethylcrotonyl-CoA) could be directly reduced to produce an isoprene alcohol followed by phosphorylation to enter the isoprenoid pathway post-MVA.",
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The Biosynthetic Incorporation of the Intact Leucine Skeleton into Sterol by the Trypanosomatid Leishmania mexicana. / Ginger, Michael L.; Chance, Michael L.; Sadler, Ian H.; Goad, L. John.

In: Journal of Biological Chemistry, Vol. 276, No. 15, 13.04.2001, p. 11674-11682.

Research output: Contribution to journalArticle

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