S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum: cloning, overexpression, purification, and biochemical characterization.
The S-adenosylhomocysteine hydrolase gene (sahase) was cloned from the Gram-positive soil bacterium Corynebacterium glutamicum (ATCC 13032) and sequenced. The sahase gene possesses an open reading frame, which consists of 1,434 nucleotides that encode 478 amino acids. The sahase gene from C. glutamicum was expressed in Escherichia coli Rosetta cells by inserting the 1,434-bp fragment downstream from the isopropyl-beta-D-thiogalactopyranoside-inducible promoter of the pET28a+ expression vector. The recombinant S-adenosylhomocysteine hydrolase from C. glutamicum (CgrSAHase) was purified efficiently by a two-step procedure, tangential ultrafiltration and affinity chromatography. The molecular weight of the CgrSAHase, estimated by gel filtration, was about 210 kDa, while sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded a relative molecular mass of 52 +/- 1 kDa. The Michaelis-Menten constants for the natural substrates of the enzyme, S-adenosylhomocysteine (SAH), adenosine, and homocysteine, were determined to be 12, 1.4, and 40 microM, respectively. The overexpression of CgrSAHase was achieved at high level (>40 mg protein/g wet cells). Because of its high capacity to synthesize SAH, this enzyme is of high biotechnological interest.
Lozada-Ramírez
J D
JD
Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Campus Espinardo, Murcia, Spain.
Martínez-Martínez
I
I
Sánchez-Ferrer
A
A
García-Carmona
F
F
D043383:Adenosylhomocysteinase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D013696:Temperature
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D043383:Adenosylhomocysteinase