Cloning, overexpression and characterization of Leishmania donovani squalene synthase.
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.
Bhargava
Prachi
P
Division of Biochemistry, Central Drug Research Institute, Lucknow, UP, India.
Kumar
Kishore
K
Chaudhaery
Shailendra S
SS
Saxena
Anil K
AK
Roy
Uma
U
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D007893:Leishmania donovani / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D015800:Protozoan Proteins; D013186:Farnesyl-Diphosphate Farnesyltransferase