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·cm-1. The Kd for binding of the enzyme and pyridoxal-5-phosphate was 0.14 ± 0.01 μm. 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 kcat 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 °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. © 2009 FEBS.