title: Arg-265: a critical residue of L.donovani cytosolic SHMT in maintaining the binding of THF and catalysis.
abstract: Serine hydroxymethyltransferase belongs to the class of pyridoxal-5-phosphate enzymes along with aspartate aminotransferase. To explore the function of residue(s) involved in binding of the carboxylate group of Tetrahydrofolic acid (THF) to L. donovani cytosolic serine hydroxymethyltransferase (LdcSHMT), the gene was cloned in pET-28(a) vector, overexpressed and purified to homogeneity. With the help of docking results of THF to the active site of protein, the key residues involved in interaction were identified. In an attempt to unravel the function of Arg265 residue involved in binding of the carboxylate group of THF, Arg-265 was mutated to Ala by site-directed mutagenesis. The Arg265Ala-LdcSHMT showed increased Km value (threefold) and decreased kcat/Km value (threefold) for H₄-folate as compared with wild type enzyme. The wild and mutant enzymes exhibited similar Km and kcat/Km values for L-allo-threonine. Unlike the wild type enzyme, mutant failed to form characteristic quinonoid intermediate and was unable to carry out the exchange of α-proton from glycine in the presence of Tetrahydrofolate. These results suggested that Arg265 residue is required for the binding of Tetrahydrofolate and may be the base that abstracts α-proton from glycine, leading to formation of quinonoid intermediate in cytosolic SHMT of L. donovani.
authors.affiliation: Division of Biochemistry, Central Drug Research Institute, CSIR, Lucknow 226031, India.
authors.forename: Shashi
authors.initials: S
authors.lastname: Gandhi
authors.affiliation: Division of Biochemistry, Central Drug Research Institute, CSIR, Lucknow 226031, India.
authors.forename: Neha
authors.initials: N
authors.lastname: Gaur
authors.affiliation: Division of Molecular and Structural Biology, Central Drug Research Institute, CSIR, Lucknow 226031, India.
authors.forename: Shagun
authors.initials: S
authors.lastname: Krishna
authors.affiliation: Division of Molecular and Structural Biology, Central Drug Research Institute, CSIR, Lucknow 226031, India.
authors.forename: Mohd Imran
authors.initials: MI
authors.lastname: Siddiqi
authors.affiliation: Division of Biochemistry, Central Drug Research Institute, CSIR, Lucknow 226031, India. Electronic address: jkscdri@yahoo.com.
authors.forename: Jitendra Kumar
authors.initials: JK
authors.lastname: Saxena
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D016054:DNA, Protozoan / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007893:Leishmania donovani / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017433:Protein Structure, Secondary; D013057:Spectrum Analysis; D013763:Tetrahydrofolates / Q000737:chemistry / Q000378:metabolism*
chemical_list: D016054:DNA, Protozoan; D013763:Tetrahydrofolates; C030371:5,6,7,8-tetrahydrofolic acid; D001120:Arginine; D012696:Glycine Hydroxymethyltransferase; D000409:Alanine
keywords: Cytosolic serinehydroxymethyltransferase; Leishmania donovani; Pyridoxal-5-phosphate; Site directed mutagenesis; Tetra hydrofolate