Genetic and Biochemical Characterization of a Phoa-Dependent Pathway for Phosphite Oxidation in Escherichia Coli

125 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2005.Detailed genetic and biochemical characterization of a pathway for phosphite oxidation in Escherichia coli is described in this thesis work. Genetic comparison of different E. coli strains revealed that a phosphite oxidation pathway is dependent on the presence of bacterial alkaline phosphatase (BAP). Transposon mutagenesis studies and biochemical isolation of the phosphite-oxidizing component(s) led to the conclusion that BAP alone is sufficient to complete this oxidation reaction. Further biochemical characterization with purified BAP verified that this enzyme oxidizes phosphite to inorganic phosphate and produces hydrogen gas. Therefore, in addition to the well characterized phosphatase activity, E. coli BAP is also a phosphite-dependent hydrogen-evolving hydrogenase. Examination of alkaline phosphatases from other organisms showed that this oxidation function is a unique property for the E. coli enzyme. However, specific mutagenesis of crucial residues for the phosphatase activity such as Ser102 and Arg166, suggests that a common activity center is required for both activities of BAP. To further explore the mechanism for this novel function of BAP, random mutagenesis was utilized to screen for mutations that have discrepant effects on the phosphatase function and the phosphite oxidation function of BAP. Mutants that can hydrolyze phosphate esters but can not oxidize phosphite were obtained. Analysis of the mutations that caused the specific phenotype revealed the majority of these mutated residues are near to the hydrolytic center of the phosphatase activity. With more than 15,000 mutants screened, no mutants that were able to oxidize phosphite but unable to hydrolyze phosphate esters were obtained. In summary, it is likely that the phosphatase function of BAP is also required to complete the phosphite oxidation reaction catalyzed by this enzyme and that the two reactions by BAP only differ in the initial step with alcohol as the leaving group for phosphatase reaction and hydride for the phosphite oxidation reaction.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD