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Applied and Environmental Microbiology, March 1999, p. 946-950, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Purification and Characterization of Gentisate 1,2-Dioxygenases from Pseudomonas alcaligenes NCIB 9867 and Pseudomonas putida NCIB 9869

Yongmei Feng,1 Hoon Eng Khoo,2 and Chit Laa Poh1,*

Department of Microbiology1 and Department of Biochemistry,2 National University of Singapore, 119260 Singapore

Received 29 June 1998/Accepted 7 December 1998

Two 3-hydroxybenzoate-inducible gentisate 1,2-dioxygenases were purified to homogeneity from Pseudomonas alcaligenes NCIB 9867 (P25X) and Pseudomonas putida NCIB 9869 (P35X), respectively. The estimated molecular mass of the purified P25X gentisate 1,2-dioxygenase was 154 kDa, with a subunit mass of 39 kDa. Its structure is deduced to be a tetramer. The pI of this enzyme was established to be 4.8 to 5.0. The subunit mass of P35X gentisate 1,2-dioxygenase was 41 kDa, and this enzyme was deduced to exist as a dimer, with a native molecular mass of about 82 kDa. The pI of P35X gentisate 1,2-dioxygenase was around 4.6 to 4.8. Both of the gentisate 1,2-dioxygenases exhibited typical saturation kinetics and had apparent Kms of 92 and 143 µM for gentisate, respectively. Broad substrate specificities were exhibited towards alkyl and halogenated gentisate analogs. Both enzymes had similar kinetic turnover characteristics for gentisate, with kcat/Km values of 44.08 × 104 s-1 M-1 for the P25X enzyme and 39.34 × 104 s-1 M-1 for the P35X enzyme. Higher kcat/Km values were expressed by both enzymes against the substituted gentisates. Significant differences were observed between the N-terminal sequences of the first 23 amino acid residues of the P25X and P35X gentisate 1,2-dioxygenases. The P25X gentisate 1,2-dioxygenase was stable between pH 5.0 and 7.5, with the optimal pH around 8.0. The P35X enzyme showed a pH stability range between 7.0 and 9.0, and the optimum pH was also 8.0. The optimal temperature for both P25X and P35X gentisate 1,2-dioxygenases was around 50°C, but the P35X enzyme was more heat stable than that from P25X. Both enzymes were strongly stimulated by 0.1 mM Fe2+ but were completely inhibited by the presence of 5 mM Cu2+. Partial inhibition of both enzymes was also observed with 5 mM Mn2+, Zn2+, and EDTA.

* Corresponding author. Mailing address: Department of Microbiology, Faculty of Medicine, National University of Singapore, 10 Kent Ridge Crescent, 119260 Singapore. Phone: 65-8743674. Fax: 65-7766872. E-mail: micpohcl{at}nus.edu.sg.

Applied and Environmental Microbiology, March 1999, p. 946-950, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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