Hsp31 of Escherichia coli K-12 is glyoxalase III

Authors

  • Krishna P. Subedi,

    1. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 335 Gwahangno, Yuseong-Gu, Daejon 305-701, Korea
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    • These authors contributed equally to this work.

  • Dongwook Choi,

    1. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 335 Gwahangno, Yuseong-Gu, Daejon 305-701, Korea
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    • These authors contributed equally to this work.

  • Insook Kim,

    1. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 335 Gwahangno, Yuseong-Gu, Daejon 305-701, Korea
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  • Bumchan Min,

    1. Analytical Science Center, Samyang Central R&D Center, 63-2, Hwaam-dong, Yuseong-gu, Daejeon 305-717, Korea
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  • Chankyu Park

    Corresponding author
    1. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 335 Gwahangno, Yuseong-Gu, Daejon 305-701, Korea
      E-mail ckpark@kaist.ac.kr; Tel. (+82) 42 350 2629; Fax (+82) 42 350 2610.
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E-mail ckpark@kaist.ac.kr; Tel. (+82) 42 350 2629; Fax (+82) 42 350 2610.

Summary

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 Km and kcat 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.

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