title: Serine hydroxymethyltransferase from Plasmodium vivax is different in substrate specificity from its homologues.
abstract: The putative gene of Plasmodium vivax serine hydroxymethyltransferase (PvSHMT; EC 2.1.2.1) was cloned and expressed in Escherichia coli. The purified enzyme was shown to be a dimeric protein with a monomeric molecular mass of 49 kDa. PvSHMT has a maximum absorption peak at 422 nm with a molar absorption coefficient of 6370 M(-1) x cm(-1). The K(d) for binding of the enzyme and pyridoxal-5-phosphate was 0.14 +/- 0.01 microM. An alternative assay for measuring the tetrahydrofolate-dependent SHMT activity based on the coupled reaction with 5,10-methylenetetrahydrofolate reductase (EC 1.5.1.20) from E. coli was developed. PvSHMT uses a ternary-complex mechanism with a k(cat) value of 0.98 +/- 0.06 s(-1) and K(m) values of 0.18 +/- 0.03 and 0.14 +/- 0.02 mM for L-serine and tetrahydrofolate, respectively. The optimum pH of the SHMT reaction was 8.0 and an Arrhenius's plot showed a transition temperature of 19 degrees C. Besides L-serine, PvSHMT forms an external aldimine complex with D-serine, L-alanine, L-threonine and glycine. PvSHMT also catalyzes the tetrahydrofolate-independent retro-aldol cleavage of 3-hydroxy amino acids. Although L-serine is a physiological substrate for SHMT in the tetrahydrofolate-dependent reaction, PvSHMT can also use D-serine. In the absence of tetrahydrofolate at high pH, PvSHMT forms an enzyme-quinonoid complex with D-serine, but not with L-serine, whereas SHMT from rabbit liver was reported to form an enzyme-quinonoid complex with L-serine. The substrate specificity difference between PvSHMT and the mammalian enzyme indicates the dissimilarity between their active sites, which could be exploited for the development of specific inhibitors against PvSHMT.
authors: Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors: Kittipat
authors: K
authors: Sopitthummakhun
authors: Somchart
authors: S
authors: Maenpuen
authors: Yongyuth
authors: Y
authors: Yuthavong
authors: Ubolsree
authors: U
authors: Leartsakulpanich
authors: Pimchai
authors: P
authors: Chaiyen
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D010966:Plasmodium vivax / Q000201:enzymology*; D015800:Protozoan Proteins / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012756:Sheep; D013053:Spectrophotometry; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D015800:Protozoan Proteins; D011994:Recombinant Proteins; D012696:Glycine Hydroxymethyltransferase