Affiliation: | (1) Laboratory of Molecular Microbiology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, 611-0011, Japan |
Abstract: | Glyoxalase I is a ubiquitous enzyme that detoxifies methylglyoxal, which is derived from glycolysis but inhibits the growth of cells from microorganisms to mammals. Here, the structural gene for glyoxalase I (glo1+) from the fission yeast Schizosaccharomyces pombe was identified. Disruption of glo1+ enhanced susceptibility to methylglyoxal, while expression of glo1+ in a glo1 mutant of Saccharomyces cerevisiae restored tolerance to this aldehyde. The glo1+ gene product was purified. The glyoxalase I of S. pombe was a monomeric enzyme with a molecular weight of 34,000 and the kcat/Km value for methylglyoxal was 4.3×107 M–1 min–1. Treatment of purified enzyme with EDTA in imidazole buffer completely abolished enzyme activity, whereas the EDTA-treated enzyme was reactivated by several divalent metal ions, such as Zn2+, Co2+, Ni2+ and Mn2+. The glyoxalase I of S. pombe exhibited fairly high thermal stability, and almost 100% activity was retained after incubating the enzyme at 60°C for 4 h. |