SEQUENCE ANALYSIS AND MOLECULAR CHARACTERIZATION OF A NITROCATECHOL DIOXYGENASE GENE FROM Pseudomonas putida

Abstract

A Pseudomonas putida capable of degrading polychlorinated biphenyl was also found to transform 4-nitrocatechol to 3-nitro-2-hydroxy-6-oxo-hexa-2,4-dienoic acid (NHODA). Crude cell extract of this bacterium exhibited an enzyme (nitrocatechol dioxygenase, Ndo) activity catalyzing this transformation. The gene encoding Ndo was cloned in E. coli. The cloned gene (ndo) expressed in E. coli had enzyme activity that degraded not only 4-nitrocatechol but also 4-chlorocatechol, 4-methylcatechol, 2,3-dihydroxybiphenyl, and 4′-chloro-2,3-dihydroxybiphenyl. Nucleotide sequence analysis of the cloned ndo exhibited an open reading frame of 939 base pairs. This sequence can encode a 313 amino acids protein of approximately molecular weight of 35 kd, which was confirmed by in vitro transcription and translation assay and SDS-PAGE analysis. A putative ribosomal binding site (GAGGAGA) was present 7 base pairs upstream from the AUG start codon and a promotor site homologous to E. coli ‘−10’ and ‘−35’ regulatory region was located at ‘−123’ and ‘−174’ area of our clone with sequences of TTGAAG and GTGACA, respectively. The deduced amino acid sequence showed 69% homology with Cdo from Burkholderia cepacia AAI. A unique insertion of 21 amino acids was found towards the N-terminal of the Ndo. Expression of ndo in strain OU83 was repressed in presence of 3-chlorobenzoic acid as judged by the decrease in the expression of ndo specific transcript.

Keywords:

• Pseudomonas putida
• Biodegradation
• Nitrocatechol 2,3-dioxygenase
• Nucleotide sequencing

ACKNOWLEDGMENTS

We express our thanks to Dr. S. Lal for helpful discussions and Dr. Y. Kumar for the synthesis of 2,3-dihydroxybiphenyl and 4-chloro-2, 3-dihydroxybiphenyl. This work was supported in part by a grant R-812827 from the Environmental Protection Agency and Oakland University research excellence fund.