Polychlorophenols are among the most pervasive organic pollutants in the United States. Several types of polychlorophenols are primarily introduced into the environment through their use as preservatives in the wood industry, as herbicides in agriculture, and as general biocides in consumer products. They persist in the environment because halogen substitution makes them recalcitrant to microbial degradation. Significantly we have identified and characterized several microbial enzymes involved in the degradation of those toxic compounds. The PIs characterization of these enzymes has revealed several novel reactions that prompt further investigation from both biochemical and structural perspectives. The PIs long-term goal is to develop a fundamental understanding of the degradation mechanisms and to define the parameters for the substrate specificity of all the participating enzymes in the biodegradation pathway of those xenobiotic pollutants. The target of the current research is focused on the comparative investigation of three monooxygenases (TcpA, TftD and PcpB) and one special dioxygenase (PcpA), all of which catalyze the dechlorination step in polychlorophenol biodegradations. In the breakdown process of those polychlorinated aromatic compounds, dechlorination is critical because partial or complete dechlorination must occur before ring-cleavage and the subsequent mineralization. However, enzymatic dechlorination has not been significantly investigated. This investigation is providing a comprehensive understanding of the unusual specificities and reactions catalyzed by those enzymes. It is contributing to the determination of the feasibility of modification of the structures of the active sites to broaden the substrate range or accelerate the kinetics. This may lead to better clean-up strategies for remediating toxic environmental sites.
Broader impacts
This research is improving the catalytic efficiency and range of substrate specificity for the biodegradative enzymes capable of bioremediation of xenobiotic pollutants. It is applying a multidisciplinary approach to the problem by combining biochemical, genetic, biophysical and mechanistic expertise. Two graduate students, including Emiliamo Sanchez, and several undergraduate students are participating in the research. High school students are also involved in the research through existing NSF-funded K-12 outreach programs at Washington State University, bringing enzymology, structural biology and environmental microbiology to the high school classroom and local Discovery Center.
Polychlorophenols, such as pentachlorophenol (PCP) and trichlorophenols (TCPs), have been widely used in numerous applications for years. Unfortunately, many of those xenobiotic compounds have displayed resistance to biodegradation and produced a serious impact on the environment and public health. Recently, degradation pathways for these recalcitrant compounds have been characterized in three bacteria; Sphingobium chlorophenolicum L-1, Cupriavidus necator JMP134, and Burkholderia phenoliruptrix (ex. B. cepacia) AC1100, providing model systems for studying biodegradation of polychlorophenols. We have identified the enzymes and regulator proteins involved in those pathways. and have developed a fundamental understanding of the participating enzymes and regulation mechanisms and their functional enhancement. The various types of dechlorinases that we have characterized display various adaptations for dechlorination reactions. Therefore it is likely that many microorganisms have quickly evolved to degrade recently released xenobiotics through mutation of the existing enzymes, acquisition of other genes from microbial community, and reassembly of new degradation pathway. Consequently, the efficiency of some of these new enzymes and pathways tend to be poor and likely still evolving, thus can be further improved through structural guided engineering of individual enzymes and rational designing of pathways. Therefore, our systematic approach dealing with three differently evolved degradation pathways provided an in-depth understanding of the specific activities for those dechlorinases and reveal the specific amino acid residues and genes that have been adopted to accommodate the new functions. The same knowledge will eventually guide us to engineer efficient enzymes and pathways for biocatalysis in biodegradation or biosynthesis.The improved enzymes and rationally designed gene clusters can then be introduced back into the microorganisms and their enhanced bioremediation capabilities will be tested for reversing the impacts of PCP and TCP. During the NSF-funding period, we have used our comprehensive approach to thoroughly characterize several novel types of enzymatic reactions that are critically important for effective bioremediation of those accumulating toxic compounds. In addition, we were able to annotate the function of many PDB-deposited proteins of unknown function in connection with our work and thus were able to establish three new enzyme groups; glutathionyl-hydroquinone reductases, p-hydroquinone 1,2-dioxygenase and 5-CHQ dehydrochlorinase. The direct societal impact from the results of this proposed research would be the scientific guidance for the remediation of a major and serious environmental pollutant in North America, PCP and TCPs. However, the source of halogenated molecules is not only anthropogenic but is also of biological or geogenic origins. Therefore our in-depth investigation of the molecular structure and reaction mechanisms of enzymes will significantly advance biochemistry, microbiology and evolutionary theories, since it directly addresses recent microbial evolutionary adaptations to anthropogenic pollutants and the critical involvement of microorganisms in the global halogen cycle. During the NSF granting period, we have introduced 14 undergraduates to the research by involving them in this research project (four were supported by this NSF; D Onofrei, K Dorance, E White, R Kinkley). Six have their names on published research (M Lam, W Alexander, A Popchock, K Lam, T Hooper, and B Danna). Mr. Walker presented his work as his Honors College thesis to WSU Honors College and received the highest honor bestowed upon Honors College undergraduate theses. 8 graduate students (A Green, R Hayes, S Belchick, B Webb, S Jun, K Lewis, A Sattler and E Sanchez) and one postdoc (A Subramanian) were participated in the research either fully or partially, and 3 PhDs and a Master were produced from it. Mr. Hayes received the excellent research award from WSU Chemistry department. Due to their excellent performance, Mr. Sanchez and Miss Green were granted the NIH Training Grant Fellowship and NIH Protein Biotech Grant respectively. PI and CoPI will continue to provide laboratory research opportunities to both graduate and undergraduate students through the grant. Participating graduate students will be introduced to essential concepts in enzyme biochemistry, X-ray crystallography, and other physical/biochemical tools, as they relate to issues in bioremediation and evolution. The same graduate students have been given the opportunity to supervise undergraduate research, which benefits both the graduate and undergraduate students. Kang (PI) has also created two new courses; "Advanced Organic Chemistry and Enzyme Reaction Mechanisms: CHEM 572" and "Chemical Biology: CHEM 370" offering since 2012.
