Characterization of Plasmodium falciparum serine hydroxymethyltransferase-A potential antimalarial target.
Serine hydroxymethyltransferase (SHMT) is a ubiquitous enzyme required for folate recycling and dTMP synthesis. A cDNA encoding Plasmodium falciparum (Pf) SHMT was expressed as a hexa-histidine tagged protein in Escherichia coli BL21-CodonPlus (DE3)-RIL. The protein was purified and the process yielded 3.6 mg protein/l cell culture. Recombinant His(6)-tagged PfSHMT exhibits a visible spectrum characteristic of pyridoxal-5'-phosphate enzyme and catalyzes the reversible conversion of l-serine and tetrahydrofolate (H(4)folate) to glycine and 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate). Steady-state kinetics study indicates that His(6)-tagged PfSHMT catalyzes the reaction by a ternary-complex mechanism. The sequence of substrate binding to the enzyme was also examined by glycine product inhibition. A striking property that is unique for His(6)-tagged PfSHMT is the ability to use D-serine as a substrate in the folate-dependent serine-glycine conversion. Kinetic data in combination with expression result support the proposal of SHMT reaction being a regulatory step for dTMP cycle. This finding suggests that PfSHMT can be a potential target for antimalarial chemotherapy.
Maenpuen
Somchart
S
Department of Biochemistry and Center for Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok, Thailand.
Sopitthummakhun
Kittipat
K
Yuthavong
Yongyuth
Y
Chaiyen
Pimchai
P
Leartsakulpanich
Ubolsree
U
D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005998:Glycine / Q000378:metabolism; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D010963:Plasmodium falciparum / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013057:Spectrum Analysis / Q000379:methods; D013763:Tetrahydrofolates / Q000378:metabolism
D018076:DNA, Complementary; D016054:DNA, Protozoan; D011993:Recombinant Fusion Proteins; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; C030371:5,6,7,8-tetrahydrofolic acid; D012694:Serine; D012696:Glycine Hydroxymethyltransferase; D005998:Glycine