title: DNA sequence determination of the TOL plasmid (pWWO) xylGFJ genes of Pseudomonas putida: implications for the evolution of aromatic catabolism.
abstract: The meta operon of the Pseudomonas putida TOL plasmid (pWWO) encodes all enzymes of a meta-cleavage pathway for the metabolism of benzoic acids to Krebs-cycle intermediates. We have determined and analysed the nucleic acid sequence of a 3442 bp region of the meta operon containing the xyl-GFJ genes whose products are involved in the post meta-ring fission transformation of catechols. Homology analysis of the xylGFJ gene products revealed evidence of biochemical relatedness, suggested enzymatic mechanisms, and permitted us to propose evolutionary events which may have generated the current variety of aromatic degradative pathways. The xylG gene, which specifies 2-hydroxymuconic semialdehyde dehydrogenase (HMSD), was found to encode a protein of 51.7 kDa. The predicted protein sequence exhibits significant homology to eukaryotic aldehyde dehydrogenases (ADHs) and to the products of two other Pseudomonas catabolic genes, i.e. xylC and alkH. Expansion of the ADH superfamily to include these prokaryotic enzymes permitted a broader analysis of functionally critical ADH residues and phylogenetic relationships among superfamily members. The importance of three regions of these enzymes previously thought to be critical to ADH activity was reinforced by this analysis. However glutamine-487, also thought to be critical, is less well conserved. The revised ADH phylogeny proposed here suggests early catabolic ADH divergence with subsequent interkingdom gene exchange. The xylF gene, which specifies 2-hydroxymuconic semialdehyde hydrolase (HMSH), was delineated by N-terminal sequence analysis of the purified gene product and is shown to encode a protein of 30.6 kDa. Homology analysis revealed sequence similarity to a chromosomally encoded serine hydrolase, especially in the region of the previously identified active-site serine residue, suggesting that HMSH may also possess a serine hydrolytic enzymatic mechanism. Likewise, the xylJ gene, which specifies 2-hydroxy-pent-2,4-dienoate hydratase (HPH), was delineated by N-terminal sequence analysis of purified HPH, and was found to encode a 23.9 kDa protein. Sequence comparisons revealed that both HMSH and HPH have analogues in the tod gene cluster, which specifies a toluene/benzene degradative pathway. Although the newly identified todF and todJ genes had been at least partially sequenced (Zylstra and Gibson, 1989), the open reading frames had not been positively identified. The presence of todJ provides strong evidence that the reactions following ring fission in the tod pathway are identical to those of the TOL pathway.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Division of Microbiology, National Research Centre for Biotechnology, Braunschweig, Germany.
authors.forename: J M
authors.initials: JM
authors.lastname: Horn
authors.forename: S
authors.initials: S
authors.lastname: Harayama
authors.forename: K N
authors.initials: KN
authors.lastname: Timmis
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics*; D000444:Aldehyde Dehydrogenase / Q000235:genetics*; D000445:Aldehyde Oxidoreductases; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D006836:Hydro-Lyases / Q000235:genetics*; D006867:Hydrolases / Q000235:genetics*; D007438:Introns; D008969:Molecular Sequence Data; D005810:Multigene Family; D009876:Operon; D010802:Phylogeny; D010957:Plasmids; D014176:Protein Biosynthesis; D011506:Proteins; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
chemical_list: D003062:Codon; D011506:Proteins; D000429:Alcohol Oxidoreductases; D000445:Aldehyde Oxidoreductases; D000444:Aldehyde Dehydrogenase; C023320:2-hydroxymuconic semialdehyde dehydrogenase; D006867:Hydrolases; C092541:2-hydroxymuconate-semialdehyde hydrolase; D006836:Hydro-Lyases; C061677:2-oxopent-4-enoate hydratase