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

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.