A novel deaminase involved in chloronitrobenzene and nitrobenzene degradation with Comamonas sp. strain CNB-1

Abstract

Comamonas sp. strain CNB-1 degrades nitrobenzene and chloronitrobenzene via the intermediates 2-aminomuconate and 2-amino-5-chloromuconate, respectively. Deamination of these two compounds results in the release of ammonia, which is used as a source of nitrogen for bacterial growth. In this study, a novel deaminase was purified from Comamonas strain CNB-1, and the gene (cnbZ) encoding this enzyme was cloned. The N-terminal sequence and peptide fingerprints of this deaminase were determined, and BLAST searches revealed no match with significant similarity to any functionally characterized proteins. The purified deaminase is a monomer (30 kDa), and its V(max) values for 2-aminomuconate and 2-amino-5-chloromuconate were 147 micromol x min(-1) x mg(-1) and 196 micromol x min(-1) x mg(-1), respectively. Its catalytic products from 2-aminomuconate and 2-amino-5-chloromuconate were 2-hydroxymuconate and 2-hydroxy-5-chloromuconate, respectively, which are different from those previously reported for the deaminases of Pseudomonas species. In the catalytic mechanism proposed, the alpha-carbon and nitrogen atoms (of both 2-aminomuconate and 2-amino-5-chloromuconate) were simultaneously attacked by a hydroxyl group and a proton, respectively. Homologs of cnbZ were identified in the genomes of Bradyrhizobium japonicum, Rhodopseudomonas palustris, and Roseiflexus sp. strain RS-1; these genes were previously annotated as encoding hypothetical proteins of unknown function. It is concluded that CnbZ represents a novel enzyme that deaminates xenobiotic compounds and/or alpha-amino acids.

FIG. 1.

Organization of the genetic fragment containing cnbZ and comparison to other genetic fragments that contain cnbZ homologs. The sequence upstream of cnbZ is shown; the predicted promoter consensus sequence is underlined, the transcription start codon is boxed, and the translational start codon is shown in italics. cnbZ and the putative arsenate resistance genes (arsRABC) are flanked by two putative transposase genes. Sequence identities of ArsC, ArsB, ArsA, and ArsR from Comamonas sp. strain CNB-1 to their counterparts from B. japonicum strain USDA 110 are 59%, 15%, 48%, and 37%, respectively. The sequence identities of Orf2 to Blr7786 and OrfJ are 44% and 41%, respectively; however, the C termini of Blr7786 and OrfJ were not found in Orf2. RBS, ribosome binding site.

FIG. 2.

Typical spectral changes during deamination of 2-aminomuconate (A) and 2-amino-5-chloromuconate (B) by purified deaminase CnbZ and proposed catalytic mechanisms. Absorption was recorded every 30 s. The amount of purified CnbZ in the reaction was 2.7 μg of protein. Below the graphs is a proposed putative mechanism of CnbZ-catalyzed deamination, which includes simultaneous attacks on the α-carbon and nitrogen atoms of 2-aminomuconate/2-amino-5-chloromuconate, respectively, by a hydroxyl group and a proton.

FIG. 3.

Analysis of cnbZ and cnbH transcription by RT-PCR. Templates used for PCR were as follows: lanes 1 and 4, cDNAs from total RNA preparation; lanes 2 and 5, total RNA preparation; lanes 3 and 6, total genomic DNA. Primers (CnbZrtPf and CnbZrtPr [Table 1]) specific for cnbZ were used for lanes 1, 2, and 3; primers (CnbHrtPf and CnbHrtPr [Table 1]) specific for cnbH were used for lanes 4, 5, and 6. Lane M, DNA markers; bands from top to bottom represent 2,500, 2,000, 1,000, 750, 500, and 250 bp.

FIG. 4.

Evolutionary dendrogram of deaminases involved in nitrobenzene and chloronitrobenzene degradation and homologs selected from results of a BLASTP search of GenBank, performed as described by Altschul et al. (1). The phylogenetic tree was generated using the neighbor-joining method with the Mega 3.1 software. *, the sequence was obtained from the SEED database (http://theseed.uchicago.edu/FIG/index.cgi). The scale at the bottom shows sequence divergence.