MAA Isomerase to Tetrachlorohydroquinone Dehalogenase: Evolution in Action

University Of Colorado At Boulder, Boulder CO

Investigators

Abstract

0077569 Copley Xenobiotic compounds released to the environment exert selective pressures on microorganisms that promote the evolution of new catabolic pathways. Recruitment of pre-existing enzymes to serve new functions is one important strategy by which microorganisms assemble new catabolic pathways. Sphingomonas chlorophenolica appears to have recruited maleylacetoacetate (MAA) isomerase to serve as a tetrachlorohydroquinone (TCHQ) dehalogenase during biodegradation of the toxic xenobiotic pesticide, pentachlorophenol. MAA isomerase catalyzes the glutathione-dependent isomerization of a double bond during the catabolism of tyrosine. TCHQ dehalogenase replaces chlorine substituents on TCHQ and trichlorohydroquinone with hydrogen atoms, a process which results in the oxidation of two molecules of glutathione to glutathione disulfide. Although these reactions are remarkably different, TCHQ dehalogenase has considerable sequence identity with known MAA isomerases in the active site region and has substantial isomerase activity. Initial efforts will be directed at a rigorous analysis of the function of the progenitor protein, including elucidation of the reaction mechanism and identification of the roles of active site residues. Subsequently, a detailed analysis of the mutations required to develop and optimize the altered activity will be carried out, with emphasis on understanding the function of each residue that is changed. Finally, in vitro evolution techniques will be used to try to evolve a more effective enzyme by alleviating the substrate inhibition that limits the catalytic ability of TCHQ dehalogenase. This work will provide new insights into the evolution of new enzymes in response to environmental toxins. It will also yield important information about the adaptation of the generic catalytic capabilities of a protein scaffold for dramatically different purposes that should provide inspiration for protein engineers interested in developing new catalysts. Finally, it will provide a gene for an improved TCHQ dehalogenase that should be useful in the engineering of an improved strain of S. chlorophenolica with an enhanced ability to degrade pentachlorophenol.