Molecular cloning and biochemical characterization of Leishmania donovani serine hydroxymethyltransferase.
Serine hydroxymethyltransferase (SHMT) catalyzes the inter conversion of serine and tetrahydrofolate (H(4)-folate) to form glycine and 5,10-methylene H(4)-folate and generates one-carbon fragments for the synthesis of nucleotides, methionine, thymidylate, choline, etc. In spite of being an indispensable enzyme of the thymidylate cycle, SHMT in Leishmania donovani remains uncharacterized. The study of L. donovani SHMT (ldSHMT) becomes important as this gene is preferentially expressed in the amastigote stage of parasite, which resides in human macrophages. Here we report cloning, expression and purification of a catalytically active ldSHMT. The homogeneity of recombinant protein was analyzed by denaturing gel electrophoresis and protein was found to be 95% pure having yield of 1mg/l. The recombinant protein is a tetramer of 216kDa as evidenced by gel filtration chromatography and uses serine and tetrahydrofolate as substrates with Km of 1.6 and 2.4mM, respectively. Further biochemical studies revealed that pH optimum of ldSHMT is 7.8 and enzyme is thermally stable up to 45 degrees C. ldSHMT was found sensitive towards denaturants as manifested by loss of enzyme activity at the concentration of 1M urea or 0.25M guanidine hydrochloride. This is the first report of purification and characterization of recombinant SHMT from any protozoan source. Studies on recombinant ldSHMT will help in evaluating this enzyme as potential drug target.
Vatsyayan
Rit
R
Biochemistry Division, Central Drug Research Institute, Lucknow 226001, India.
Roy
Uma
U
D000818:Animals; D003001:Cloning, Molecular; D015870:Gene Expression; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry / Q000145:classification / Q000235:genetics*; D007893:Leishmania donovani / Q000201:enzymology* / Q000378:metabolism; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D012696:Glycine Hydroxymethyltransferase