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

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 degrees C for 10 min, but extracts containing HabA were inactivated when they were heated at temperatures above 60 degrees 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.

FIG. 1

Initial steps in the enzyme-catalyzed pathway of nitrobenzene degradation by P. pseudoalcaligenes JS45 contrasted with the chemical Bamberger rearrangement of hydroxylaminobenzene to 4-aminophenol (not observed in JS45).

FIG. 2

Functional and restriction map of the 8-kb region of the JS45 chromosome encoding the two HAB mutase isozymes. habA, gene encoding HAB mutase A; habB, gene encoding HAB mutase B; IR, tnpR, and tnpA′, regions with similarity to the inverted repeat, resolvase gene, and truncated transposase gene of transposon Tn5501, respectively; tnpA, open reading frame that exhibits low amino acid sequence similarity to the transposase of IS801. The antibiotic cassettes and the positions of their insertions in the habA-minus and habB-minus strains are indicated. AI, PinAI; K, KpnI; P, PstI; S, SalI; Sh, SphI; Sp, SpeI; X, XbaI. Asterisks indicate restriction sites that were mutated as described in Materials and Methods.

FIG. 3

ClustalX alignment of the amino acid sequences of the two isozymes of HAB mutase of JS45 (HabA and HabB) and of the product of gene Rv3078 from M. tuberculosis. The asterisks below the alignment indicate amino acids that are conserved in all three sequences.

FIG. 4

RT-PCR of RNA prepared from strain JS45 grown in LB or in mineral medium supplemented with nitrobenzene. Lanes 1 and 12, molecular weight markers; lane 2, RNA from LB-grown cells, habA primers, RT present; lane 3, RNA from LB-grown cells, habA primers, RT absent; lane 4, RNA from nitrobenzene-grown cells, habA primers, RT present; lane 5, RNA from nitrobenzene-grown cells, habA primers, RT absent; lane 6, PCR control containing JS45 DNA and habA primers; lane 7, PCR control containing JS45 DNA and habB primers; lane 8, RNA from nitrobenzene-grown cells, habB primers, RT present; lane 9, RNA from LB-grown cells, habB primers, RT present; lane 10, RNA from nitrobenzene-grown cells, habB primers, RT absent; lane 11, RNA from LB-grown cells, habB primers, RT absent.

FIG. 5

Thermostabilities of HAB mutases. Symbols: ×, HabA; ▵, HabB; ○, JS45. Prior to the enzyme assay, the crude extracts were preincubated at the temperatures indicated for 10 min and then placed on ice. The 100% level of activity corresponded to 0.263 μmol · min⁻¹ · mg of protein⁻¹ for HabA, 3.15 μmol · min⁻¹ · mg of protein⁻¹ for HabB, and 6.24 μmol · min⁻¹ · mg of protein⁻¹ for JS45.