Sequence analysis and initial characterization of two isozymes of hydroxylaminobenzene mutase from Pseudomonas pseudoalcaligenes JS45

Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene by a partially red uctive pathway in which the intermediate hydroxylaminobenzene is enzymatica lly rearranged to 2-aminophenol by hydroxylaminobenzene mutase (HAB mutase) , The properties of the enzyme, the reaction mechanism, and the evolutionar y 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, w ere 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 H abB are 44% identical. The HAB mutase specific activities in crude extracts of Escherichia coil clones synthesizing either HabA or HabB were similar t o the specific activities of extracts of strain JS45 grown on nitrobenzene. HAB mutase activity in E. coli extracts containing HabB withstood heating at 85 degrees C for 10 min, but extracts containing HabA were inactivated w hen they were heated at temperatures above 60 degrees C. HAB mutase activit y in extracts of P. pseudoalcaligenes JS45 grown on nitrobenzene exhibited intermediate temperature stability, Although both the habA gene and the hab B gene conferred HAB mutase activity when they were separately cloned and e xpressed 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 gr ew on nitrobenzene. Gene Rv3078 of Mycobacterium tuberculosis H37Rv encodes a protein whose deduced amino acid sequence is 52% identical to the HabB a mino acid sequence. E. coli containing M. tuberculosis gene Rv3078 cloned i nto pUC18 exhibited low levels of HAB mutase activity. Sequences that exhib it similarity to transposable element sequences are present between habA an d habB, as well as downstream of habB, which suggests that horizontal gene transfer resulted in acquisition of one or both of the hab genes.