Cloning, overexpression and characterization of Leishmania donovani squalene synthase

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.

Biochemical characterization of the water‐soluble squalene synthase from Methylococcus capsulatus and the functional analyses of its two DXXD(E)D motifs and the highly conserved aromatic amino acid residues

“…Recombinant SQSs truncated at the N-and/or C-terminus region are functionally expressed in E. coli and prepared as a soluble and active form by using a detergent [6][7][8][9][10][11][12][13]. All of the SQSs identified previously require a detergent for their high activity, with CHAPS and Tween 80 having been used mainly for activity enhancement [6][7][8][9][10][11][12][13]. Recently, Lee and Poulter [14] reported the first characterization of a prokaryotic SQS from T. elongatus BP-1 (teSQS; Fig.…”

“…Until now, many SQSs have been cloned from eukaryotes and functionally expressed in E. coli as a truncated and soluble form [5][6][7][8][9][10][11][12][13]. However, investigations of prokaryotic SQSs are very limited, and only one example from Thermosynechococcus elongatus BP-1 has been reported [14].…”

Developments in Diagnosis and Antileishmanial Drugs

“…A number of enzymes in the ergosterol biosynthetic pathway have been investigated as potential drug targets for these organisms and have shown great promise. Thus, C14-demethylase, sterol 24-methyltransferase, 3-hydroxy-3-methylglutaryl CoA reductase, squalene epoxide, squalene synthase, and farnesyl pyrophosphate synthase have been studied both individually and in combination, with varying degrees of success [103, 104]. Ergosterol biosynthesis inhibitors with potent in vitro activity and special pharmacokinetic properties in mammals can induce radical parasitological cure in animal models of several forms of leishmaniasis [105].…”

The genetic toolbox forLeishmaniaparasites

“…51,52 Overexpression of certain genes can also help to understand their biological function as the phenotypic effects caused by the investigated protein will be amplified. 53,54 A novel system utilizing artificial linear episome-based structures shows promise for expressing large quantities of target proteins in L. tarentolae for the purpose of protein purification. 55,56 A unique challenge to the development of regulated expression systems is posed by the unusual lack of transcriptional control in Leishmania parasites.…”