Characterization of anthranilate and benzoate 1,2-dioxygenase from Acinetobacter sp. strain ADP1
Beharry, Zanna Maria
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Many oxygen activating enzymes that contain nonheme mononuclear iron active sites catalyze metabolically important reactions that are environmentally and medically significant. In an effort to meet demands for economical and environmentally-friendly oxidation processes and for specific oxidation products, biocatalytic routes are receiving much attention. The Rieske dioxygenases (RDO) catalyze the initial reaction in the microbial degradation of aromatic environmental pollutants. The products of the RDO reaction are also potentially useful intermediates for natural product syntheses. While more than three dozen distinct RDOs have been identified differing in their substrate specificity, few have been isolated due to instability or low expression. The aerobic soil bacterium Acinetobacter sp. strain ADP1 (ADP1) is able to utilize the aromatic compounds anthranilate and benzoate as sole carbon sources. The enzyme systems responsible for the initial degradation of these compounds are the chromosomallyencoded Rieske dioxygenases anthranilate 1,2-dioxygenase (AntDO) and benzoate 1,2- dioxygenase (BenDO). In this dissertation, a combination of molecular biological, biochemical and spectroscopic techniques are used to characterize the in vitro substrate specificity of AntDO and BenDO and demonstrate the importance of a highly conserved aspartate residue throughout the RDOs in catalysis. The results presented show that both AntDO and BenDO can dihydroxylate both anthranilate and benzoate to form the expected in vivo products, contrary to previous results that determined AntDO and BenDO are specific for anthranilate and benzoate in vivo, respectively. In addition, the chromosomally-encoded AntDO and BenDO can dihydroxylate a number of substituted benzoates, which conflicts with the notion that plasmid-encoded RDOs are able to dihydroxylate a much wider range of aromatic compounds than their chromosomallyencoded counterparts.