Cloning, overexpression and characterization of Leishmania donovani squalene synthase

FEMS Microbiol Lett. 2010 Oct;311(1):82-92. doi: 10.1111/j.1574-6968.2010.02071.x. Epub 2010 Aug 16.

Abstract

Squalene synthase (SSN, EC 2.5.1.21), a major enzyme in the sterol biosynthetic pathway, catalyses an unusual head-to-head reductive dimerization of two molecules of farnesyl-pyrophosphate (FPP) in a two-step reaction to form squalene. FPP serves as a metabolic intermediate in the formation of sterols, dolichols, ubiquinones and farnesylated proteins. Here, we report cloning, expression and purification of a catalytically active recombinant squalene synthase of Leishmania donovani (LdSSN). The pH and temperature optima of LdSSN were 7.4 and 37°C, respectively. Biochemical studies revealed that the K(m) and V(max) for the substrate FPP were 3.8 μM and 0.59 nM min(-1) mg(-1) and for NADPH were 43.23 μM and 0.56 nM min(-1) mg(-1). LdSSN was found to be sensitive towards denaturants as manifested by a loss of enzyme activity at the concentration of 1 M urea or 0.25 M guanidine hydrochloride. Zaragozic acid A, a potent inhibitor of mammalian SSN, was also a competitive inhibitor of recombinant LdSSN, with a K(i) of 74 nM. This is the first report on the purification and characterization of full-length recombinant SSN from L. donovani. Studies on recombinant LdSSN will help in evaluating this enzyme as a potential drug target for visceral leishmaniasis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cloning, Molecular*
  • Enzyme Stability
  • Farnesyl-Diphosphate Farnesyltransferase / chemistry*
  • Farnesyl-Diphosphate Farnesyltransferase / genetics*
  • Farnesyl-Diphosphate Farnesyltransferase / isolation & purification
  • Farnesyl-Diphosphate Farnesyltransferase / metabolism
  • Gene Expression*
  • Kinetics
  • Leishmania donovani / chemistry
  • Leishmania donovani / classification
  • Leishmania donovani / enzymology*
  • Leishmania donovani / genetics
  • Molecular Sequence Data
  • Phylogeny
  • Protozoan Proteins / chemistry*
  • Protozoan Proteins / genetics*
  • Protozoan Proteins / isolation & purification
  • Protozoan Proteins / metabolism
  • Sequence Alignment

Substances

  • Protozoan Proteins
  • Farnesyl-Diphosphate Farnesyltransferase

Associated data

  • GENBANK/AM229310