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Applied and Environmental Microbiology, March 2004, p. 1804-1810, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1804-1810.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Construction of Chimeric Catechol 2,3-Dioxygenase Exhibiting Improved Activity against the Suicide Inhibitor 4-Methylcatechol

Akiko Okuta, Kouhei Ohnishi,^* and Shigeaki Harayama

Marine Biotechnology Institute, Heita, Kamaishi, Iwate 026-0001, Japan

Received 7 July 2003/ Accepted 16 December 2003

Catechol 2,3-dioxygenase (C23O; EC 1.3.11.2), exemplified by XylE and NahH, catalyzes the ring cleavage of catechol and some substituted catechols. C23O is inactivated at an appreciable rate during the ring cleavage of 4-methylcatechol due to the oxidation of the Fe(II) cofactor to Fe(III). In this study, a C23O exhibiting improved activity against 4-methylcatechol was isolated. To isolate this C23O, diverse C23O gene sequences were PCR amplified from DNA which had been isolated from mixed cultures of phenol-degrading bacteria and subcloned in the middle of a known C23O gene sequence (xylE or nahH) to construct a library of chimeric C23O genes. These chimeric C23O genes were then introduced into Pseudomonas putida possessing some of the toluene catabolic genes (xylXYZLGFJQKJI). When a C23O gene (e.g., xylE) is introduced into this strain, the transformants cannot generally grow on p-toluate because 4-methylcatechol, a metabolite of p-toluate, is a substrate as well as a suicide inhibitor of C23O. However, a transformant of this strain capable of growing on p-toluate was isolated, and a chimeric C23O (named NY8) in this transformant was characterized. The rate of enzyme inactivation by 4-methylcatechol was lower in NY8 than in XylE. Furthermore, the rate of the reactivation of inactive C23O in a solution containing Fe(II) and ascorbic acid was higher in NY8 than in XylE. These properties of NY8 might allow the efficient metabolism of 4-methylcatechol and thus allow host cells to grow on p-toluate.

Present address: Japan Biological Information Research Center, Kouto-ku, Tokyo 135-0064, Japan.

Present address: Research Institute of Molecular Genetics, Kochi University, Nankoku, Kochi 783-8502, Japan.