Genetic and biochemical studies of aromatic dioxygenase substrate specificity in Acinetobacter sp. strain ADP1
Eby, Donald matthew
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This dissertation describes the characterization of anthranilate 1,2- dioxygenase. This enzyme and other dioxygenases play an essential role in aromatic compound catabolism by Acinetobacter sp. strain ADP1. Two separate enzymes that constitute anthranilate 1,2-dioxygenase were expressed in Escherichia coli and purified to homogeneity: a reductase and a terminal dioxygenase. The electron-transferring and catalytic metal centers were identified and characterized. Furthermore, the size and quantity of anthranilate 1,2-dioxygenase subunits were analyzed, and deduced amino acid sequences were used to evaluate evolutionary relatedness among bacterial aromatic ring hydroxylating dioxygenases. Comprehensive activity measurements were completed for anthranilate 1,2-dioxygenase and a homolog, the benzoate 1,2- dioxygenase from ADP1. In additional studies, an ADP1 derived mutant was used to measure the efficiency of generating random mutations and introducing them into the chromosomal catA gene that encodes catechol 1,2-dioxygenase. The conditions for mutagenesis were optimized using two different PCR-based approaches for incorporating random mutations. The optimization procedure took advantage of the natural transformability of ADP1-derived strains and the ability to select mutants directly in which mutations has generated non-functional catechol 1,2-dioxygenase enzymes. These studies lay the foundation for continuing efforts to alter the substrate specificity of catechol 1,2-dioxygenase by random mutation. This dissertation also reports investigations aimed at developing an Acinetobacter host system for the synthesis of dioxygenases encoded by genes from diverse microorganisms. An expression vector was constructed containing an engineered Acinetobacter promoter to facilitate high-level gene expression without the need for a transcriptional activator. To test the system, the wild-type pcaHG genes, encoding protocatechuate 3,4-dioxygenase, were deleted from the ADP1 chromosome. These genes were then expressed in trans from the vector, and the effects were determined with regards to growth on protocatechuate or p-hydroxybenzoate as the sole carbon source. Furthermore, the expression vector was used to demonstrate that the pcaHG genes from a marine Roseobacter isolate could complement the effect of the engineered deletion in the ADP1-derived host strain. Collectively, these studies constitute the beginning steps into engineering ADP1 as a host for altering the substrate specificity and catalytic activity of aromatic dioxygenases in the effort to generate novel growth phenotypes.