Structure-Guided Studies of Bacterial Competition Mechanisms
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Microorganisms have evolved a stunning array of strategies for nutrient competition, ranging from concerted effort of antibiotic release to kill off competing species, to evolving complex enzymatic pathways that are capable of scavenging nutrients from sources not utilizable by other organisms. The carbon-phosphorous (C-P) lyase pathway is a survival mechanism that is activated during phosphate limitation in certain species of bacteria and enables cleavage of the extremely stable C-P bond in order to obtain phosphorous from organophosphonates. The structure and biochemical characterization of PhnP, a critical accessory protein from C-P lyase pathway of E. coli is presented in this thesis. The structure of PhnP revealed a conserved metal-dependent hydrolase active site with two Mn2+ ions, and another unique mononuclear Zn2+ site that appears to have a structural role. A non-physiological ligand that fortuitously co-crystallized with the enzyme provided insights into the catalytic features of the active site. We were able to demonstrate hydrolytic activity towards a number of phosphodiesterase substrates, and propose a plausible physiological role for PhnP. These results contribute to deciphering the mechanism of phosphonate utilization, which would allow design of bioremediation programs to remove toxic phsophonates from the environment. Antibiotic production is another mechanism for resource competition. Structural characterization of CmlS, a halogenase from the chloramphenicol biosynthesis pathway of S. venezuelae is presented. The crystal structure revealed a novel covalent modification of its FAD cofactor, which was confirmed through ESI-MS and chemical denaturation studies. The unique C-terminal domain, active site architecture, and the position of the C-terminus suggest that halogenation mechanism of CmlS may differ from the currently proposed mechanism for structurally related halogenases. This work provides early steps towards understanding mechanisms of enzymatic halogenations, which is of great scientific, as well as pharmaceutical interest.