This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Werwath, J.
Right arrow Articles by Wittich, R.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Werwath, J.
Right arrow Articles by Wittich, R.-M.

 Previous Article  |  Next Article 

Journal of Bacteriology, August 1998, p. 4171-4176, Vol. 180, No. 16
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Biochemical and Genetic Characterization of a Gentisate 1,2-Dioxygenase from Sphingomonas sp. Strain RW5

Jörn Werwath, Hans-Adolf Arfmann, Dietmar H. Pieper, Kenneth N. Timmis, and Rolf-Michael Wittich*

Division of Microbiology, GBF-National Research Centre for Biotechnology, D-38124 Braunschweig, Germany

Received 23 March 1998/Accepted 4 June 1998

A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC 1.13.11.4), gtdA, from Sphingomonas sp. strain RW5 was cloned and sequenced. The gtdA gene encodes a 350-amino-acid polypeptide with a predicted size of 38.85 kDa. Comparison of the gtdA gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no significant homology except for a low similarity (27%) to the 1-hydroxy-2-naphthoate dioxygenase (phdI) of the phenanthrene degradation in Nocardioides sp. strain KP7 (T. Iwabuchi and S. Harayama, J. Bacteriol. 179:6488-6494, 1997). This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases. The gene was subcloned and hyperexpressed in E. coli. The resulting product was purified to homogeneity and partially characterized. Under denaturing conditions, the polypeptide exhibited an approximate size of 38.5 kDa and migrated on gel filtration as a species with a molecular mass of 177 kDa. The enzyme thus appears to be a homotetrameric protein. The purified enzyme stoichiometrically converted gentisate to maleylpyruvate, which was identified by gas chromatography-mass spectrometry analysis as its methyl ester. Values of affinity constants (Km) and specificity constants (Kcat/Km) of the enzyme were determined to be 15 µM and 511 s-1 M-1 × 104 for gentisate and 754 µM and 20 s-1 M-1 × 104 for 3,6-dichlorogentisate. Three further open reading frames (ORFs) were found downstream of gtdA. The deduced amino acid sequence of ORF 2 showed homology to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomerase superfamily and glutathione reductase superfamily, respectively.

* Corresponding author. Mailing address: Division of Microbiology, GBF-National Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. Phone: (49) 531 6181 557. Fax: (49) 531 6181 411. E-mail: wittich{at}gbf.de.

Journal of Bacteriology, August 1998, p. 4171-4176, Vol. 180, No. 16
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Deveryshetty, J., Phale, P. S. (2009). Biodegradation of phenanthrene by Pseudomonas sp. strain PPD: purification and characterization of 1-hydroxy-2-naphthoic acid dioxygenase. Microbiology 155: 3083-3091 [Abstract] [Full Text]  
  • Feng, J., Che, Y., Milse, J., Yin, Y.-J., Liu, L., Ruckert, C., Shen, X.-H., Qi, S.-W., Kalinowski, J., Liu, S.-J. (2006). The Gene ncgl2918 Encodes a Novel Maleylpyruvate Isomerase That Needs Mycothiol as Cofactor and Links Mycothiol Biosynthesis and Gentisate Assimilation in Corynebacterium glutamicum. J. Biol. Chem. 281: 10778-10785 [Abstract] [Full Text]  
  • Tan, C. L., Yeo, C. C., Khoo, H. E., Poh, C. L. (2005). Replacement of Tyrosine 181 by Phenylalanine in Gentisate 1,2-Dioxygenase I from Pseudomonas alcaligenes NCIMB 9867 Enhances Catalytic Activities. J. Bacteriol. 187: 7543-7545 [Abstract] [Full Text]  
  • Shen, X.-H., Jiang, C.-Y., Huang, Y., Liu, Z.-P., Liu, S.-J. (2005). Functional Identification of Novel Genes Involved in the Glutathione-Independent Gentisate Pathway in Corynebacterium glutamicum. Appl. Environ. Microbiol. 71: 3442-3452 [Abstract] [Full Text]  
  • Hintner, J.-P., Reemtsma, T., Stolz, A. (2004). Biochemical and Molecular Characterization of a Ring Fission Dioxygenase with the Ability to Oxidize (Substituted) Salicylate(s) from Pseudaminobacter salicylatoxidans. J. Biol. Chem. 279: 37250-37260 [Abstract] [Full Text]  
  • Ishiyama, D., Vujaklija, D., Davies, J. (2004). Novel Pathway of Salicylate Degradation by Streptomyces sp. Strain WA46. Appl. Environ. Microbiol. 70: 1297-1306 [Abstract] [Full Text]  
  • Zhuang, Z., Song, F., Takami, H., Dunaway-Mariano, D. (2004). The BH1999 Protein of Bacillus halodurans C-125 Is Gentisyl-Coenzyme A Thioesterase. J. Bacteriol. 186: 393-399 [Abstract] [Full Text]  
  • Diaz, E., Ferrandez, A., Prieto, M. A., Garcia, J. L. (2001). Biodegradation of Aromatic Compounds by Escherichia coli. Microbiol. Mol. Biol. Rev. 65: 523-569 [Abstract] [Full Text]  
  • Hintner, J.-P., Lechner, C., Riegert, U., Kuhm, A. E., Storm, T., Reemtsma, T., Stolz, A. (2001). Direct Ring Fission of Salicylate by a Salicylate 1,2-Dioxygenase Activity from Pseudaminobacter salicylatoxidans. J. Bacteriol. 183: 6936-6942 [Abstract] [Full Text]  
  • Zhou, N.-Y., Fuenmayor, S. L., Williams, P. A. (2001). nag Genes of Ralstonia (Formerly Pseudomonas) sp. Strain U2 Encoding Enzymes for Gentisate Catabolism. J. Bacteriol. 183: 700-708 [Abstract] [Full Text]  
  • Dunwell, J. M., Khuri, S., Gane, P. J. (2000). Microbial Relatives of the Seed Storage Proteins of Higher Plants: Conservation of Structure and Diversification of Function during Evolution of the Cupin Superfamily. Microbiol. Mol. Biol. Rev. 64: 153-179 [Abstract] [Full Text]  
  • Miyauchi, K., Adachi, Y., Nagata, Y., Takagi, M. (1999). Cloning and Sequencing of a Novel meta-Cleavage Dioxygenase Gene Whose Product Is Involved in Degradation of gamma -Hexachlorocyclohexane in Sphingomonas paucimobilis. J. Bacteriol. 181: 6712-6719 [Abstract] [Full Text]  
  • Bartels, F., Backhaus, S., Moore, E. R. B., Timmis, K. N., Hofer, B. (1999). Occurrence and expression of glutathione-S-transferase-encoding bphK genes in Burkholderia sp. strain LB400 and other biphenyl-utilizing bacteria. Microbiology 145: 2821-2834 [Abstract] [Full Text]  
  • Armengaud, J., Timmis, K. N., Wittich, R.-M. (1999). A Functional 4-Hydroxysalicylate/Hydroxyquinol Degradative Pathway Gene Cluster Is Linked to the Initial Dibenzo-p-Dioxin Pathway Genes in Sphingomonas sp. Strain RW1. J. Bacteriol. 181: 3452-3461 [Abstract] [Full Text]  
  • Feng, Y., Khoo, H. E., Poh, C. L. (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 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»