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Enzymes for applications in bioremedation : a site-directed mutagenesis study of second coordination sphere residues in PcpA
DaRosa, Paul A.
May 10, 2010
Chlorinated xenobiotic compounds such as pentachlorophenol are destructive to the environment and are toxic to most organisms. The removal of these chemicals is difficult due to their intrinsic properties. The stability and limited biodegradability of chlorinated aromatic compounds make these chemicals particularly challenging targets for bioremediation. Degradation pathways of chlorinated arenes tend to be inefficient due to dead end metabolites and bottlenecks. To increase the rate of biodegradation and remove bottlenecks, creation of hybrid strains through patchwork assembly, pathway engineering, or protein engineering may be necessary. This can only be achieved if the enzymes in these pathways are well characterized. In this study, site-directed mutagenesis was performed on Sphingobium chlorophenolicum 2,6-dichlorohydroquinone 1,2-dioxygenase (PcpA). PcpA is a nonheme Fe(II)-containing enzyme that oxidatively cleaves 2,6-dichlorohydroquinone in the degradation pathway of pentachlorophenol and has potential applications in bioremediation. The second coordination sphere residues, H49 and R259, were mutated to various amino acids to study the role of these residues in the enzymes’ structure and catalysis. R259 is proposed to have an important structural role in pcpA, while H49 is proposed to have an acid/base role in the oxidative ring cleavage of 2,6-dichlorohydroquinone by PcpA. The information obtained from these mutations offers a starting point for further studies of substrate specificity and the catalytic mechanism of PcpA. This information may be critical to PcpA’s possible bioremediation applications.
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