DNA sequence determination of the TOL plasmid (pWW0) 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)

Molecular characterization of the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli W: engineering a mobile aromatic degradative cluster

“…The gene hpaE encodes a protein of 53,011 Da that presents all the motifs that characterize the aldehyde dehydrogenase superfamily (23). The HpaE protein shows a 98.6% identity to its putative counterpart, 5-carboxymethyl-2-hydroxy-muconic semialdehyde dehydrogenase of E. coli C (16,44), and about 40% identity to other meta-cleavage pathway aldehyde dehydrogenases, such as the DmpC and XylG proteins from the phenol pathway of Pseudomonas sp.…”

“…The HpaE protein shows a 98.6% identity to its putative counterpart, 5-carboxymethyl-2-hydroxy-muconic semialdehyde dehydrogenase of E. coli C (16,44), and about 40% identity to other meta-cleavage pathway aldehyde dehydrogenases, such as the DmpC and XylG proteins from the phenol pathway of Pseudomonas sp. strain CF600 and the TOL pathway of P. putida, respectively (23,44,47).…”

“…Only a few aromatic catabolic pathways have been completely sequenced so far (23,26,47,53), but only the one presented here proceeds exclusively via a dehydrogenase/decarboxylase branch. Moreover, an overall analysis of this cluster reveals that six proteins, HpaR, HpaB, HpaC, HpaD, HpaF, and HpaI, do not present any similarity to functionally analogous proteins of other degradative pathways, suggesting that they evolved from a different origin.…”

What We Can Deduce about Metabolism in the Moderate Halophile Chromohalobacter Salexigens from its Genomic Sequence

“…2; Nakazawa and Yokota, 1973). For the meta-pathway, the genome was queried with ORFs present on the Pseudomonas putida TOL plasmid pWWO (Horn et al, 1991), and for the orthopathway, the genome was searched against the enzymes from Acinetobacter sp. (Barbe et al, 2004).…”

“…The usual disclaimers about inferences from genomic sequence are in order: that all of the annotations that we are assigning are provisional and await confirmation by biochemical or functional genetic analyses, and that the predictions of the functions of the C. salexigens gene products are only as valid as the accuracy of the assignment of the function of matching proteins in other organisms. was based on the chapter by Fraenkel (1996), the tricarboxylic acid (TCA) cycle by Cronan and LaPorte (1996), peripheral carbon source metabolism by Lin (1996) and Gottschalk (1985), and aromatic compound metabolism by Horn et al (1991) and Nakazawa and Yokota (1973). For the tracing of the aromatic degradative pathways we also made extensive use of the University of Minnesota Biocatalysis/Biodegradation Database (http://umbbd.ahc.umn.edu).…”