Author: Rehman, Abdul
Cytochrome P450cam (CYP101A1) from the soil bacterium Pseudomonas putida oxidizes camphor regio- and stereoselectively at the 5-position, to give 5-exo¬-hydroxycamphor. In order to alter the substrate range of P450cam, it has to be mutated. Previously, we have randomly mutated P450cam and selected seven mutants on the bicyclic polychlorinated pollutant endosulfan (ES). Endosulfan is a pesticide and is a persistent organic pollutant (POP). Endosulfan diol (ES diol), which is the major hydrolysis product of endosulfan in the environment, is also persistent in the environment, along with endosulfan itself. Here, we describe the activity of the P450cam mutants towards biodegradation of endosulfan diol. The P450cam mutants convert these substrates to substituted ortho-quinones, which we detected using 4-aminoantipyrine (4-AAP) in the assays. Here, we have studied the dehalogenation of endosulfan diol catalyzed by the endosulfan – selected P450cam mutants, using in vitro kinetics, chloride release assays and 13C labeled endosulfan diol. ES7 (V247F/D297N/K314E) was found to be the most active mutant, significantly more active than the wild type (WT) towards biodegradation of ES diol. On average ~ 5.2 Cl- ions are released per aromatic product detected upon turnover of ES diol. Based on these findings, we propose a mechanism that begins with the epoxidation of the ES-substrate's double bond on the norbornene system, proceeds with elimination of six chloride ions and loss of the bridge as CO2, to furnish the ortho-quinone. The monoterpene β-phellandrene is released by certain species of pine when placed under stress. Due to the limited supply of β-phellandrene available from natural sources, here we describe a short synthesis of racemic β-phellandrene from readily available β-pinene. Furthermore, oxidized monoterpenes are known to be released by plants and to function as attractants or repellents of insects, so it is of interest to find ways of selectively oxidizing β-phellandrene. The compound was found to be a substrate for WT-P450cam and the ES7 mutant. In in vitro assays with the cytochrome P450cam, β-phellandrene was hydroxylated.
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Thesis advisor: Plettner, Erika
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