Inorganic phosphate (Pi) plays a critical role in many biological structures and processes. However, Pi typically occurs at low concentrations, particularly in marine environments. In comparison, naturally occurring organophosphonates, which are characterized by a stable carbon-phosphorus (CP) bond, are frequently present at higher concentrations. Accordingly, bacteria have evolved different mechanisms for cleaving the CP bond of organophosphonates to liberate Pi for metabolic use. Two prominent enzyme pathways for catabolic cleavage of a CP bond are examined in this thesis. The first, called CP-lyase, is encoded by the phn operon that consists of 14 genes (phnCDEFGHIJKLMNOP). CP-lyase has long been of interest for its ability to degrade a wide array of organophosphonates through a homolytic CP bond cleaving reaction. A soluble protein complex consisting of PhnGHIJK was isolated from E. coli, suggesting that protein-protein interactions are important for CP bond cleavage. Intermediates of organophosphonate catabolism by E. coli CP-lyase were also detected and isolated, including -D-ribosyl-1,2-cyclic phosphate and N-acetylated aminoalkylphosphonates, 2-N-acetamidoethylphosphonate and 5’-phospho--D-ribosyl-1’-alkylphosphonates. The former compound was shown to be converted by the phosphodiesterase PhnP to -D-ribosyl-1-phosphate. It was also shown that PhnO is an aminoalkylphosphonate N-acetyl transferase and that N-acetylation by this enzyme is necessary for CP bond cleavage of 1-aminoalklyphosphonates. These results demonstrated that in addition to forming protein complexes, CP-lyase also comprises a catabolic pathway, with ribosylation of organophosphonates playing a key part in setting up the CP bond cleaving reaction. The second pathway examined in this thesis is comprised of marine bacterial enzymes PhnY and PhnZ and is specific for 2-aminoethylphosphonate. PhnY was shown to be an -ketoglutarate / Fe(II) dependent dioxygenase that hydroxylates the -carbon of 2-aminoethylphosphonate to form (R)-2-amino-1-hydroxyethylphosphonate. PhnZ was shown to be a novel Fe(II) dependent oxygenase that converts (R)-2-amino-1-hydroxyethylphosphonate to glycine and Pi. Site directed mutagenesis, kinetic analysis, reactions with substrate analogues, and X-ray crystallography examined the roles of active site residues and the di-iron active site. Additionally, a unique induced-fit mechanism was discovered which appears to synchronize substrate binding with activation of molecular oxygen. Overall these results show that PhnZ represents a new mechanism for catabolic cleavage of a CP bond. / Thesis (Ph.D, Chemistry) -- Queen's University, 2013-09-13 16:24:17.261