Sequence Analysis and Initial Characterization of Two Isozymes of Hydroxylaminobenzene Mutase from Pseudomonas pseudoalcaligenes JS45
- 1 July 2000
- journal article
- research article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 66 (7), 2965-2971
- https://doi.org/10.1128/aem.66.7.2965-2971.2000
Abstract
Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene by a partially reductive pathway in which the intermediate hydroxylaminobenzene is enzymatically rearranged to 2-aminophenol by hydroxylaminobenzene mutase (HAB mutase). The properties of the enzyme, the reaction mechanism, and the evolutionary origin of the gene(s) encoding the enzyme are unknown. In this study, two open reading frames ( habA and habB ), each encoding an HAB mutase enzyme, were cloned from a P. pseudoalcaligenes JS45 genomic library and sequenced. The open reading frames encoding HabA and HabB are separated by 2.5 kb and are divergently transcribed. The deduced amino acid sequences of HabA and HabB are 44% identical. The HAB mutase specific activities in crude extracts of Escherichia coli clones synthesizing either HabA or HabB were similar to the specific activities of extracts of strain JS45 grown on nitrobenzene. HAB mutase activity in E. coli extracts containing HabB withstood heating at 85°C for 10 min, but extracts containing HabA were inactivated when they were heated at temperatures above 60°C. HAB mutase activity in extracts of P. pseudoalcaligenes JS45 grown on nitrobenzene exhibited intermediate temperature stability. Although both the habA gene and the habB gene conferred HAB mutase activity when they were separately cloned and expressed in E. coli , reverse transcriptase PCR analysis indicated that only habA is transcribed in P. pseudoalcaligenes JS45. A mutant strain derived from strain JS45 in which the habA gene was disrupted was unable to grow on nitrobenzene, which provided physiological evidence that HabA is involved in the degradation of nitrobenzene. A strain in which habB was disrupted grew on nitrobenzene. Gene Rv3078 of Mycobacterium tuberculosis H37Rv encodes a protein whose deduced amino acid sequence is 52% identical to the HabB amino acid sequence. E. coli containing M. tuberculosis gene Rv3078 cloned into pUC18 exhibited low levels of HAB mutase activity. Sequences that exhibit similarity to transposable element sequences are present between habA and habB , as well as downstream of habB , which suggests that horizontal gene transfer resulted in acquisition of one or both of the hab genes.Keywords
This publication has 31 references indexed in Scilit:
- Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequenceNature, 1998
- Novel Genes Encoding 2-Aminophenol 1,6-Dioxygenase fromPseudomonas Species AP-3 Growing on 2-Aminophenol and Catalytic Properties of the Purified EnzymeJournal of Biological Chemistry, 1997
- CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choiceNucleic Acids Research, 1994
- Alcohol dehydrogenase-dependent reduction of 2-nitrosofluorene and rearrangement of N-hydroxy-2-aminofluoreneBiochemistry, 1993
- An improved suicide vector for construction of chromosomal insertion mutations in bacteriaGene, 1992
- IS801, an insertion sequence element isolated from Pseudomonas syringae pathovar phaseolicolaMolecular Microbiology, 1991
- Studies on transformation of Escherichia coli with plasmidsJournal of Molecular Biology, 1983
- A simple method for displaying the hydropathic character of a proteinJournal of Molecular Biology, 1982
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976
- Ueber das PhenylhydroxylaminEuropean Journal of Inorganic Chemistry, 1894