title: Hsp31 of Escherichia coli K-12 is glyoxalase III.
abstract: 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.
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 335 Gwahangno, Yuseong-Gu, Daejon 305-701, Korea.
authors.forename: Krishna P
authors.initials: KP
authors.lastname: Subedi
authors.forename: Dongwook
authors.initials: D
authors.lastname: Choi
authors.forename: Insook
authors.initials: I
authors.lastname: Kim
authors.forename: Bumchan
authors.initials: B
authors.lastname: Min
authors.forename: Chankyu
authors.initials: C
authors.lastname: Park
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D001711:Biotransformation; D002413:Cations, Divalent / Q000378:metabolism; D004252:DNA Mutational Analysis; D004791:Enzyme Inhibitors / Q000378:metabolism; D048168:Escherichia coli K12 / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007773:Lactates / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011765:Pyruvaldehyde / Q000378:metabolism; D013696:Temperature
chemical_list: D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D007773:Lactates; D008670:Metals; D018832:Molecular Chaperones; C469996:hchA protein, E coli; D011765:Pyruvaldehyde; D000445:Aldehyde Oxidoreductases; C091855:glyoxalase III