Cloning, expression, and characterization of a novel methylglyoxal synthase from Thermus sp. strain GH5.
A gene encoding methylglyoxal synthase from Thermus sp. GH5 (TMGS) was cloned, sequenced, overexpressed, and purified by Q-Sepharose. The TMGS gene was composed of 399 bp which encoded a polypeptide of 132 amino acids with a molecular mass of 14.3 kDa. The K (m) and k (cat) values of TMGS were 0.56 mM and 325 (s(-1)), respectively. The enzyme exhibited its optimum activity at pH 6 and 75 degrees C. Comparing the amino acid sequences and Hill coefficients of Escherichia coli MGS and TMGS revealed that the loss of Arg 150 in TMGS has caused a decrease in the cooperativity between the enzyme subunits in the presence of phosphate as an allosteric inhibitor. Gel filtration experiments showed that TMGS is a hexameric enzyme, and its quaternary structure did not change in the presence of phosphate.
Pazhang
Mohammad
M
Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran.
Khajeh
Khosro
K
Asghari
S Mohsen
SM
Falahati
Hanieh
H
Naderi-Manesh
Hossein
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019757:Carbon-Oxygen Lyases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D016415:Sequence Alignment; D013824:Thermus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D019757:Carbon-Oxygen Lyases; C021578:methylglyoxal synthase