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Identification of a novel gentisate 1,2-dioxygenase from Silicibacter pomeroyi

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Abstract

A 1,125-bp long ORF encoding a novel gentisate 1,2-dioxygenase with two-domain bicupins was cloned from Silicibacter pomeroyi DSS-3 and expressed in Escherichia coli. The resulting product was purified to homogeneity and partially characterized. Non-reductive SDS-PAGE and gel filtration showed that the active recombinant gentisate 1,2-dioxygenase had an estimated molecular mass of 132 kDa, and reductive SDS-PAGE indicated an approximate size of 45 kDa. The enzyme thus appears to be a homotrimeric protein. This is in contrast to the homotetrameric or dimeric protein of the gentisate 1,2-dioxygenases that have been characterized thus far. The K m and K cat/K m for gentisate were 12 μM and 653 × 104 M−1 s−1; the pI was 4.6–4.8. It was optimally active at 40°C and pH 8.0.

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References

  • Adams MA, Singh VK, Keller BO, Jia Z (2006) Structural and biochemical characterization of gentisate 1,2-dioxygenase from Escherichia coli O157:H7. Mol Microbiol 61:1469–1484

    Article  PubMed  CAS  Google Scholar 

  • Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  PubMed  CAS  Google Scholar 

  • Chua CH, Feng YM, Yeo CC, Khoo HE, Poh CL (2001) Identification of amino acid residues essential for catalytic activity of gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIB 9867. FEMS Microbiol Lett 204:141–146

    Article  PubMed  CAS  Google Scholar 

  • Clewley JP (1995) Macintosh sequence analysis software. DNAstar’s LaserGene. Mol Biotechnol 3:221–224

    PubMed  CAS  Google Scholar 

  • Crawford RL, Hutton SW, Chapman PJ (1975) Purification and properties of gentisate 1,2-dioxygenase from Moraxella osloensis. J Bacteriol 121:794–799

    PubMed  CAS  Google Scholar 

  • Feng YM, Khoo HE, Poh CL (1999) Purification and characterization of gentisate 1,2-dioxygenases from Pseudomonas alcaligenes NCIB 9867 and Pseudomonas putida NCIB 9869. Appl Environ Microbiol 65:946–950

    PubMed  CAS  Google Scholar 

  • Fu W, Oriel P (1998) Gentisate 1,2-dioxygenase from Haloferax sp. D1227. Extremophiles 2:439–446

    Article  PubMed  CAS  Google Scholar 

  • Gao X, Tan CL, Yeo CC, Poh CL (2005) Molecular and biochemical characterization of the xlnD-encoded 3-hydroxybenzoate 6-hydroxylase involved in the degradation of 2,5-xylenol via the gentisate pathway in Pseudomonas alcaligenes NCIMB 9867. J Bacteriol 187:7696–7702

    Article  PubMed  CAS  Google Scholar 

  • Harpel MR, Lipscomb JD (1990) Gentisate 1,2-dioxygenase from Pseudomonas. Purification, characterization, and comparison of the enzymes from Pseudomonas testosteroni and Pseudomonas acidovorans. J Biol Chem 265:6301–6311

    PubMed  CAS  Google Scholar 

  • Jeanmougin F, Thompson JD, Gouy M, Higgins DG, Gibson TJ (1998) Multiple sequence alignment with Clustal X. Trends Biochem Sci 23:403–405

    Article  PubMed  CAS  Google Scholar 

  • Jones DC, Cooper RA (1990) Catabolism of 3-hydroxybenzoate by the gentisate pathway in Klebsiella pneumoniae M5a1. Arch Microbiol 54:489–495

    Article  Google Scholar 

  • Kloetzel MC, Abadir BY (1955) Synthetic analogs of cortical hormones. Part II. 3-substituted α-2,5-trihydroxyacetophenone derivatives. J Am Chem Soc 77:3823–3826

    Article  CAS  Google Scholar 

  • Kumar SK, Tamura K, Nei M (2004) MEGA3: an integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163

    Article  PubMed  CAS  Google Scholar 

  • Lack L (1959) The enzymic oxidation of gentisic acid. Biochim Biophys Acta 34:117–123

    Article  PubMed  CAS  Google Scholar 

  • Luo S, Liu DQ, Liu H, Zhou NY (2006) Site-directed mutagenesis of gentisate 1,2-dioxygenases from Klebsiella pneumoniae M5a1 and Ralstonia sp. strain U2. Microbiol Res 161:138–144

    Article  PubMed  CAS  Google Scholar 

  • Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31:3381–3385

    Article  PubMed  CAS  Google Scholar 

  • Shen XH, Jiang CY, Huang Y, Liu ZP, Liu SJ (2005) Functional identification of novel genes involved in the glutathione-independent gentisate pathway in Corynebacterium glutamicum. Appl Environ Microbiol 71:3442–3452

    Article  PubMed  CAS  Google Scholar 

  • Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130

    Article  PubMed  CAS  Google Scholar 

  • Suarez M, Ferrer E, Martin M (1996) Purification and biochemical characterization of gentisate 1,2-dioxygenase from Klebsiella pneumoniae M5a1. FEMS Microbiol Lett 143:89–95

    Article  PubMed  CAS  Google Scholar 

  • Werwath J, Arfmann HA, Pieper DH, Timmis KN, Wittich RM (1998) Biochemical and genetic characterization of a gentisate 1,2-dioxygenase from Sphingomonas sp. strain RW5. J Bacteriol 180:4171–4176

    PubMed  CAS  Google Scholar 

  • Wheelis ML, Palleroni NJ, Stanier RY (1967) The metabolism of aromatic acids by Pseudomonas testosteroni and Pseudomonas acidovorans. Arch Mikrobiol 59:302–314

    Article  PubMed  CAS  Google Scholar 

  • Zhou NY, Fuenmayor SL, Williams PA (2001) nag genes of Ralstonia (formerly Pseudomonas) sp. strain U2 encoding enzymes for gentisate catabolism. J Bacteriol 183:700–708

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by Postdoctoral Science Foundation of China (Grant No. 2005038032). We also thank F. Xiang, Z. Li and L.Y. Zhou for their assistance during this work.

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Authors and Affiliations

  1. College of Environmental Sciences, Peking University, Beijing, 100871, P.R. China

    Dongqi Liu, Tingting Zhu, Li Fan & Jinren Ni

  2. Shenzhen Graduate School, Peking University, Shenzhen, 518055, P.R. China

    Dongqi Liu, Tingting Zhu, Li Fan, Junming Quan & Hongchun Guo

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  1. Dongqi Liu
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  2. Tingting Zhu
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  3. Li Fan
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Correspondence to Dongqi Liu.

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Liu, D., Zhu, T., Fan, L. et al. Identification of a novel gentisate 1,2-dioxygenase from Silicibacter pomeroyi . Biotechnol Lett 29, 1529–1535 (2007). https://doi.org/10.1007/s10529-007-9421-7

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  • DOI: https://doi.org/10.1007/s10529-007-9421-7

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