cDNA cloning, overexpression in Escherichia coli, purification and characterization of sheep liver cytosolic serine hydroxymethyltransferase.
A sheep liver cDNA clone for the cytosolic serine hydroxymethyltransferase (SHMT) was isolated and its nucleotide sequence determined. The full-length cDNA of SHMT was placed under the control of T7 promoter in pET-3C plasmid and expressed in Escherichia coli. The overexpressed enzyme, present predominantly in the soluble fraction, was catalytically active. The recombinant SHMT was purified to homogeneity with a yield of 10 mg/l bacterial culture. The recombinant enzyme was capable of carrying out tetrahydrofolate-dependent and tetrahydrofolate-independent reactions as effectively as the native enzyme. The Km values for serine (1 mM) and tetrahydrofolate (0.82 mM) were similar to those of the native enzyme. The recombinant enzyme had a characteristic visible spectrum indicative of the presence of pyridoxal 5'-phosphate as an internal aldimine. The apoenzyme obtained upon removal of the cofactor was inactive and could be reconstituted by the addition of pyridoxal 5'-phosphate demonstrating that the recombinant SHMT was functionally very similar to the native SHMT. This overexpression of eukaryotic tetrameric SHMT in E. coli and the purification and characterization of the recombinant enzyme should thus allow studies on the role of specific amino acids and domains in the activity of the enzyme.
Jagath-Reddy
J
J
Department of Biochemistry, Indian Institute of Science, Bangalore.
Ganesan
K
K
Savithri
H S
HS
Datta
A
A
Rao
N A
NA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary; D004926:Escherichia coli; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics* / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D012756:Sheep
D018076:DNA, Complementary; D012696:Glycine Hydroxymethyltransferase