title: Cloning and structural analysis of Mycobacterium leprae serine hydroxymethyltransferase.
abstract: Serine hydroxymethyltransferase (SHMT) plays a key role in cell physiology as it participates in the different interconversion pathway of folate coenzymes, provides almost exclusively folate one carbon fragments for the biosynthesis of a variety of end products. For the first time, Mycobacterium leprae glyA gene, encodes the enzyme serine hydroxymethyltransferase, has been cloned in Escherichia coli, over-expressed and purified the protein product (mlSHMT) for folding and stability studies under various denaturating conditions. The recombinant mlSHMT exists as homo-dimer of molecular mass about 90 kDa under physiological conditions . The studies on catalytic properties of mlSHMT show that the enzyme catalyzes the H(4)-folate dependent retro-aldol cleavage of L-serine, however, D-alanine dependent transaminase activity was absent in the enzyme. Further analysis of the enzyme kinetics for hydroxymethyltransferase reaction for mlSHMT demonstrates a comparable K(m) value for L-serine to SHMTs from other sources but significantly lower catalytic efficiency (k(cat)/K(m)). The mlSHMT is resistant to alkaline denaturation and exist as apo-dimer up to pH 10.5. Urea and guanidinium chloride induces dissociation of mlSHMT dimer into monomer at low denaturant concentrations, and leads to loss of enzymatic activity.
authors.affiliation: Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226001, India. sarita.sharma05@gmail.com
authors.forename: Sarita
authors.initials: S
authors.lastname: Sharma
authors.forename: Vinod
authors.initials: V
authors.lastname: Bhakuni
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability; D012696:Glycine Hydroxymethyltransferase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009166:Mycobacterium leprae / Q000201:enzymology*; D011487:Protein Conformation; D017510:Protein Folding
chemical_list: D012696:Glycine Hydroxymethyltransferase