Subunit interactions within the carbon-phosphorus lyase complex from escherichia coli

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.