A family 13 thioesterase isolated from an activated sludge metagenome: Insights into aromatic compounds metabolism

Abstract

Activated sludge is produced during the treatment of sewage and industrial wastewaters. Its diverse chemical composition allows growth of a large collection of microbial phylotypes with very different physiologic and metabolic profiles. Thus, activated sludge is considered as an excellent environment to discover novel enzymes through functional metagenomics, especially activities related with degradation of environmental pollutants. Metagenomic DNA was isolated and purified from an activated sludge sample. Metagenomic libraries were subsequently constructed in Escherichia coli. Using tributyrin hydrolysis, a screening by functional analysis was conducted and a clone that showed esterase activity was isolated. Blastx analysis of the sequence of the cloned DNA revealed, among others, an ORF that encodes a putative thioesterase with 47-64% identity to GenBank CDS reported genes, similar to those in the hotdog fold thioesterase superfamily. On the basis of its amino acid similarity and its homology-modelled structure we deduced that this gene encodes an enzyme (ThYest_ar) that belongs to family TE13, with a preference for aryl-CoA substrates and a novel catalytic residue constellation. Plasmid retransformation in E. coli confirmed the clone's phenotype, and functional complementation of a paaI E. coli mutant showed preference for phenylacetate over chlorobenzene as a carbon source. This work suggests a role for TE13 family thioesterases in swimming and degradation approaches for phenyl acetic acid. Proteins 2017; 85:1222-1237. © 2017 Wiley Periodicals, Inc.

Polystyrene Degradation by Exiguobacterium sp. RIT 594: Preliminary Evidence for a Pathway Containing an Atypical Oxygenase

“…Some of the genes in this cluster are the phenylacetate-CoA oxygenase subunits PaaI (RDB32562.1) and PaaJ (RDB32563.1), the 1,2-phenylacetyl-CoA epoxidase subunits A (RDB32560.1) and B (RDB32561.1), the phenylacetate-CoA ligase (RDB32559.1), and a family 13 thioesterase (RDB32558.1). These are part of the phenylacetyl-CoA catabolon [66], which includes family 13 thioesterase [67]. An unusual putative ether reductase of the EthD family is also present in the gene cluster, suggesting a pathway different from the one involving an oxepin-CoA intermediate.…”

Molecular biology interventions for activity improvement and production of industrial enzymes

“…Furthermore, 25 families of thioesterases have been identified to play active role biocatalysis of protein, lipids, and aromatic compounds metabolism. Metagenomic approach has been used by the researchers to isolate novel thioesterases belonging to family 10 13, from activated sludge samples, which play important role in biocatalysis of phenylacetic acid and 4-chlorobenzene, both compounds associated with environmental pollution (Sanchez-Reyez et al, 2017).…”

Thioesterase enzyme families: Functions, structures, and mechanisms

“…New TE substrate specificities have been determined: as examples, (a) in TE4, a preference toward short chain fatty acids was observed in ACOT 18 ; (b) RpaL, a TesB-like TE4 enzyme from Rhodopseudomonas palustris, was found to be active on aromatic and long and short aliphatic molecules bound to CoA 19 ; (c) in TE6, YciA enzymes from Methylobacterium extorquens were shown to be hydrolyze ethylmalonyl-CoA for dicarboxylic acid production 20 ; and, (d) aryl-CoA substrate specificity was observed for enzymes in TE13. 21 More TEs have been identified since we first classified TEs into families, some which form part of existing families. As examples, (a) guanosine diphosphate regulation TEs from Neisseria meningitidis appear in TE6 22 ; (b) acyllipid thioesterase from Arabidopsis thaliana in TE9 23 ; (c) methylketone synthases, 24 which were originally characterized from tomato prior to the ThYme database, have also been found in Solanum melongena and Glycine max and form part of TE9 25,26 ; (d) Shewanella oneidensis YbgC, which was found to primarily hydrolyze short chain acyl-CoA thioesters, also forms part of TE9 27 ; (e) BorB, required for borrelidin biosynthesis, is a member of TE18 28 ; and (f) the Isochrysis galbana thioesterase/ carboxylesterase (IgTeCe) is in TE21.…”