title: Five phosphonate operon gene products as components of a multi-subunit complex of the carbon-phosphorus lyase pathway.
abstract: Organophosphonate utilization by Escherichia coli requires the 14 cistrons of the phnCDEFGHIJKLMNOP operon, of which the carbon-phosphorus lyase has been postulated to consist of the seven polypeptides specified by phnG to phnM. A 5,660-bp DNA fragment encompassing phnGHIJKLM is cloned, followed by expression in E. coli and purification of Phn-polypeptides. PhnG, PhnH, PhnI, PhnJ, and PhnK copurify as a protein complex by ion-exchange, size-exclusion, and affinity chromatography. The five polypeptides also comigrate in native-PAGE. Cross-linking of the purified protein complex reveals a close proximity of PhnG, PhnI, PhnJ, and PhnK, as these subunits disappear concomitant with the formation of large cross-linked protein complexes. Two molecular forms are identified, a major form of molecular mass of approximately 260 kDa, a minor form of approximately 640 kDa. The stoichiometry of the protein complex is suggested to be PhnG(4)H(2)I(2)J(2)K. Deletion of individual phn genes reveals that a strain harboring plasmid-borne phnGHIJ produces a protein complex consisting of PhnG, PhnH, PhnI, and PhnJ, whereas a strain harboring plasmid-borne phnGIJK produces a protein complex consisting of PhnG and PhnI. We conclude that phnGHIJK specify a soluble multisubunit protein complex essential for organophosphonate utilization.
authors.affiliation: Department of Molecular Biology, Aarhus University, 10 Gustav Wieds Vej, DK-8000 Aarhus, Denmark.
authors.forename: Bjarne
authors.initials: B
authors.lastname: Jochimsen
authors.forename: Signe
authors.initials: S
authors.lastname: Lolle
authors.forename: Fern R
authors.initials: FR
authors.lastname: McSorley
authors.forename: Mariah
authors.initials: M
authors.lastname: Nabi
authors.forename: Jens
authors.initials: J
authors.lastname: Stougaard
authors.forename: David L
authors.initials: DL
authors.lastname: Zechel
authors.forename: Bjarne
authors.initials: B
authors.lastname: Hove-Jensen
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005796:Genes; D005798:Genes, Bacterial; D008190:Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D008954:Models, Biological; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009876:Operon; D063065:Organophosphonates / Q000378:metabolism*; D021122:Protein Subunits; D018085:Regulon; D017384:Sequence Deletion
chemical_list: D004269:DNA, Bacterial; D009097:Multienzyme Complexes; D063065:Organophosphonates; D021122:Protein Subunits; D008190:Lyases; C051328:carbon-phosphorus lyase