Hsp31 encoded by hchA is known as a heat-inducible molecular chaperone. Although structure studies revealed that Hsp31 has a putative catalytic triad consisting of Asp-214, His-186 and Cys-185, its enzymatic function, besides weak amino-peptidase activity, is still unknown. We found that Hsp31 displays glyoxalase activity that catalyses the conversion of methylglyoxal (MG) to d-lactate without an additional cofactor. The glyoxalase activity was completely abolished in the hchA-deficient strain, confirming the relationship between the hchA gene and its enzymatic activity in vivo. Hsp31 exhibits Michaelis-Menten kinetics for substrates MG with K(m) and k(cat) of 1.43±0.12 mM and 156.9±5.5 min⁻¹ respectively. The highest glyoxalase activity was found at 35-40 °C and pH of 6.0-8.0, and the activity was significantly inhibited by Cu²⁺, Fe³⁺ and Zn²⁺. Mutagenesis studies based on our evaluation of conserved catalytic residues revealed that the Cys-185 and Glu-77 were essential for catalysis, whereas His-186 was less crucial for enzymatic function, although it participates in the catalytic process. The stationary-phase Escherichia coli cells became more susceptible to MG when hchA was deleted, which was complemented by an expression of plasmid-encoded hchA. Furthermore, an accumulation of intracellular MG was observed in hchA-deficient strains.
© 2011 Blackwell Publishing Ltd.