title: Identification and characterization of an acyl-CoA dehydrogenase from Pseudomonas putida KT2440 that shows preference towards medium to long chain length fatty acids.
abstract: Diverse and elaborate pathways for nutrient utilization, as well as mechanisms to combat unfavourable nutrient conditions make Pseudomonas putida KT2440 a versatile micro-organism able to occupy a range of ecological niches. The fatty acid degradation pathway of P. putida is complex and correlated with biopolymer medium chain length polyhydroxyalkanoate (mcl-PHA) biosynthesis. Little is known about the second step of fatty acid degradation (β-oxidation) in this strain. In silico analysis of its genome sequence revealed 21 putative acyl-CoA dehydrogenases (ACADs), four of which were functionally characterized through mutagenesis studies. Four mutants with insertionally inactivated ACADs (PP_1893, PP_2039, PP_2048 and PP_2437) grew and accumulated mcl-PHA on a range of fatty acids as the sole source of carbon and energy. Their ability to grow and accumulate biopolymer was differentially negatively affected on various fatty acids, in comparison to the wild-type strain. Inactive PP_2437 exhibited a pattern of reduced growth and PHA accumulation when fatty acids with lengths of 10 to 14 carbon chains were used as substrates. Recombinant expression and biochemical characterization of the purified protein allowed functional annotation in P. putida KT2440 as an ACAD showing clear preference for dodecanoyl-CoA ester as a substrate and optimum activity at 30 °C and pH 6.5-7.
authors.affiliation: School of Biomolecular and Biomedical Sciences, University College Dublin, Ardmore House, Belfield, Dublin 4, Ireland.
authors.forename: Maciej W
authors.initials: MW
authors.lastname: Guzik
authors.affiliation: School of Biomolecular and Biomedical Sciences, University College Dublin, Ardmore House, Belfield, Dublin 4, Ireland.
authors.forename: Tanja
authors.initials: T
authors.lastname: Narancic
authors.affiliation: Institute for Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11000 Belgrade, Serbia.
authors.forename: Tatjana
authors.initials: T
authors.lastname: Ilic-Tomic
authors.affiliation: Institute for Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11000 Belgrade, Serbia.
authors.forename: Sandra
authors.initials: S
authors.lastname: Vojnovic
authors.affiliation: School of Biomolecular and Biomedical Sciences, University College Dublin, Ardmore House, Belfield, Dublin 4, Ireland.
authors.forename: Shane T
authors.initials: ST
authors.lastname: Kenny
authors.affiliation: School of Biomolecular and Biomedical Sciences, University College Dublin, Ardmore House, Belfield, Dublin 4, Ireland.
authors.forename: William T
authors.initials: WT
authors.lastname: Casey
authors.affiliation: School of Chemical and Bioprocess Engineering, Engineering and Materials Science Centre, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Gearoid F
authors.initials: GF
authors.lastname: Duane
authors.affiliation: School of Chemical and Bioprocess Engineering, Engineering and Materials Science Centre, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Eoin
authors.initials: E
authors.lastname: Casey
authors.affiliation: School of Physics, Trinity College Dublin, Dublin 2, Ireland.
authors.forename: Trevor
authors.initials: T
authors.lastname: Woods
authors.affiliation: Centre for Research, Adoptive Nano Structures and Nanodevices, Trinity College Dublin, Dublin 2, Ireland.|School of Physics, Trinity College Dublin, Dublin 2, Ireland.
authors.forename: Ramesh Padamati
authors.initials: RP
authors.lastname: Babu
authors.affiliation: Institute for Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11000 Belgrade, Serbia.
authors.forename: Jasmina
authors.initials: J
authors.lastname: Nikodinovic-Runic
authors.affiliation: School of Biomolecular and Biomedical Sciences, University College Dublin, Ardmore House, Belfield, Dublin 4, Ireland.
authors.forename: Kevin E
authors.initials: KE
authors.lastname: O'Connor
mesh_terms: D042964:Acyl-CoA Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016958:Pseudomonas putida / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005227:Fatty Acids; D042964:Acyl-CoA Dehydrogenase