Evolution of Enzymes Required for Biodegradation of Pentachlorophenol in Sphingobium Chlorophenolicum
Final rept. 1 Aug 2003-31 Jan 2007
COLORADO UNIV AT BOULDER OFFICE OF CONTRACTS AND GRANTS
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Pentachlorophenol PCP is a highly toxic pesticide first introduced into the environment in 1936. PCP can be mineralized by Sphingobium chlorophenolicum. However, biodegradation is slow and the bacterium cannot tolerate high levels of PCP. Flux through the pathway is limited by the first enzyme, PCP hydroxylase, which converts PCP to tetrachlorobenzoquinone TCBQ very slowly. Furthermore, it catalyzes a futile cycling reaction in which C4a-hydroperoxyflavin at the active site decays to produce H2O2 without hydroxylating the substrate, thus wasting NADPH and producing a toxic by-product. The enzyme also hydroxylates tetrachlorohydroquinone, a downstream metabolite. This reaction generates TCBQ, effectively reversing the normal direction of the pathway. Finally, TCBQ forms an adduct with the flavin and inactivates the enzyme. The enzyme has clearly not yet evolved to be an effective catalyst for hydroxylation of PCP. Improvements in the ability of Sphingobium chlorophenolicum to degrade PCP can be generated using genome shuffling. We created several strains that degraded PCP faster and tolerated higher concentrations of PCP than the wild type strain. Notably, none of the strains contained an improved version of PCP hydroxylase, suggesting that mutations that affect other processes can have an important effect on the efficiency of the degradation pathway.
- Agricultural Chemistry
- Pesticides Pollution and Control