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 A 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.