title: 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.
authors.affiliation: Centro de Investigación en Dinámica Celular, IICBA, Universidad Autónoma del Estado de Morelos (UAEM), Colonia Chamilpa, CP, 62209, Cuernavaca, Morelos, Mexico.|Centro de Investigación en Biotecnología UAEM, CP 62209, Cuernavaca, Morelos, Mexico.
authors.forename: Ayixon
authors.initials: A
authors.lastname: Sánchez-Reyez
authors.affiliation: Centro de Investigación en Dinámica Celular, IICBA, Universidad Autónoma del Estado de Morelos (UAEM), Colonia Chamilpa, CP, 62209, Cuernavaca, Morelos, Mexico.
authors.forename: Ramón Alberto
authors.initials: RA
authors.lastname: Batista-García
authors.affiliation: Centro de Investigación en Dinámica Celular, IICBA, Universidad Autónoma del Estado de Morelos (UAEM), Colonia Chamilpa, CP, 62209, Cuernavaca, Morelos, Mexico.
authors.forename: Gilberto
authors.initials: G
authors.lastname: Valdés-García
authors.affiliation: Instituto de Biotecnología. Universidad Nacional Autónoma de México, CP 62210, Cuernavaca, Morelos, Mexico.
authors.forename: Ernesto
authors.initials: E
authors.lastname: Ortiz
authors.affiliation: Instituto de Biotecnología. Universidad Nacional Autónoma de México, CP 62210, Cuernavaca, Morelos, Mexico.
authors.forename: Lucía
authors.initials: L
authors.lastname: Perezgasga
authors.affiliation: Centro de Investigación en Biotecnología UAEM, CP 62209, Cuernavaca, Morelos, Mexico.
authors.forename: Andrés
authors.initials: A
authors.lastname: Zárate-Romero
authors.affiliation: Centro de Investigación en Dinámica Celular, IICBA, Universidad Autónoma del Estado de Morelos (UAEM), Colonia Chamilpa, CP, 62209, Cuernavaca, Morelos, Mexico.
authors.forename: Nina
authors.initials: N
authors.lastname: Pastor
authors.affiliation: Centro de Investigación en Biotecnología UAEM, CP 62209, Cuernavaca, Morelos, Mexico.
authors.forename: Jorge Luis
authors.identifier: 0000-0002-1860-8175
authors.initials: JL
authors.lastname: Folch-Mallol
mesh_terms: D000595:Amino Acid Sequence; D001673:Biodegradation, Environmental; D002722:Chlorobenzenes / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D005816:Genetic Complementation Test; D006801:Humans; D007700:Kinetics; D054892:Metagenome; D056186:Metagenomics; D016366:Open Reading Frames; D010648:Phenylacetates / Q000737:chemistry* / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012722:Sewage / Q000382:microbiology*; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D002722:Chlorobenzenes; D010648:Phenylacetates; D011994:Recombinant Proteins; D012722:Sewage; D013869:Thiolester Hydrolases; C025136:phenylacetic acid; C031294:chlorobenzene
keywords: functional high-throughput screenings; genetic complementation; metagenomics; molecular grafting; swimming motility phenotypes