Overexpression and characterization of dimeric and tetrameric forms of recombinant serine hydroxymethyltransferase from Bacillus stearothermophilus.
Serine hydroxymethyltransferase (SHMT), a pyridoxal-5' -phosphate (PLP) dependent enzyme catalyzes the interconversion of L-Ser and Gly using tetrahydrofolate as a substrate. The gene encoding for SHMT was amplified by PCR from genomic DNA of Bacillus stearothermophilus and the PCR product was cloned and overexpressed in Escherichia coli. The purified recombinant enzyme was isolated as a mixture of dimer (90%) and tetramer (10%). This is the first report demonstrating the existence of SHMT as a dimer and tetramer in the same organism. The specific activities at 37 C of the dimeric and tetrameric forms were 6 7 U/mg and 4 1 U/mg, respectively. The purified dimer was extremely thermostable with a T(m) of 85 degrees C in the presence of PLP and L-Ser. The temperature optimum of the dimer was 80 degrees C with a specific activity of 32 4 U/mg at this temperature. The enzyme catalyzed tetrahydrofolate-independent reactions at a slower rate compared to the tetrahydrofolate-dependent retro-aldol cleavage of L-Ser. The interaction with substrates and their analogues indicated that the orientation of PLP ring of B. stearothermophilus SHMT was probably different from sheep liver cytosolic recombinant SHMT (scSHMT).
Jala
Venkatakrishna R
VR
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
Prakash
V
V
Rao
N Appaji
NA
Savithri
H S
HS
D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D012696:Glycine Hydroxymethyltransferase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D008024:Ligands; D016133:Polymerase Chain Reaction; D020836:Protein Structure, Quaternary; D013696:Temperature
D008024:Ligands; D012696:Glycine Hydroxymethyltransferase