title: Subunit Interactions within the Carbon-Phosphorus Lyase Complex from Escherichia coli.
abstract: Phosphonates are a large class of organophosphorus compounds with a characteristic carbon-phosphorus bond. The genes responsible for phosphonate utilization in Gram-negative bacteria are arranged in an operon of 14 genes. The carbon-phosphorus lyase complex, encoded by the genes phnGHIJKLM, catalyzes the cleavage of the stable carbon-phosphorus bond of organophosphonates to the corresponding hydrocarbon and inorganic phosphate. Recently, complexes of this enzyme containing five subunits (PhnG-H-I-J-K), four subunits (PhnG-H-I-J), and two subunits (PhnG-I) were purified after expression in Escherichia coli ( Proc. Natl. Acad. Sci., U. S. A. 2011 , 108 , 11393 ). Here we demonstrated using mass spectrometry, ultracentrifugation, and chemical cross-linking experiments that these complexes are formed from a PhnG2I2 core that is further elaborated by the addition of two copies each of PhnH and PhnJ to generate PhnG2H2I2J2. This complex adds an additional subunit of PhnK to form PhnG2H2I2J2K. Chemical cross-linking of the five-component complex demonstrated that PhnJ physically interacts with both PhnG and PhnI. We were unable to demonstrate the interaction of PhnH or PhnK with any other subunits by chemical cross-linking. Hydrogen-deuterium exchange was utilized to probe for alterations in the dynamic properties of individual subunits within the various complexes. Significant regions of PhnG become less accessible to hydrogen/deuterium exchange from solvent within the PhnG2I2 complex compared with PhnG alone. Specific regions of PhnI exhibited significant differences in the H/D exchange rates in PhnG2I2 and PhnG2H2I2J2K.
authors.forename: Zhongjie
authors.initials: Z
authors.lastname: Ren
authors.forename: Soumya
authors.initials: S
authors.lastname: Ranganathan
authors.forename: Nathanael F
authors.initials: NF
authors.lastname: Zinnel
authors.forename: William K
authors.initials: WK
authors.lastname: Russell
authors.forename: David H
authors.initials: DH
authors.lastname: Russell
authors.forename: Frank M
authors.initials: FM
authors.lastname: Raushel
mesh_terms: D000595:Amino Acid Sequence; D041961:Deuterium Exchange Measurement; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D008190:Lyases / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D021122:Protein Subunits; D008190:Lyases; C051328:carbon-phosphorus lyase