Several PCPs, such as 2,4,5- and 2,4,6-trichlorophenol (TCP), 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 chloride substitution makes them recalcitrant to microbial degradation. The high levels of EDTA in natural waters are also due to its extensive usage, such as in industrial cleaning, in detergent and in phytoremediation to mobilize heavy metals. In the environment, EDTA occurs in metal-EDTA complexes, most of which are highly recalcitrant. Recently, several enzymes from soil microorganisms have been characterized, which degrade 2,4,5-TCP, 2,4,6-TCP and EDTA. Our on-going research is essential for improving the catalytic efficiency and substrate range of critical enzymes involved in biodegradation of recalcitrant pollutants, targeting effective bioremediation.

In the breakdown process of PCPs, the most critical step is the oxidation at the para-position catalyzed by monooxygenases, because partial or complete dechlorination must occur before ring-cleaving dioxygenases are able to open aromatic rings. We will focus on two critical monooxygenases; 2,4,5-TCP 4-monooxygenase (TftD) and 2,4,6-TCP 4-monooxygenase (TcpA). Both TftD and TcpA share a high degree of sequence similarity (65% identity) and use 2,4,6-TCP as a substrate; however, the two enzymes produce different end-products. TcpA catalyzes both oxidative and hydrolytic reactions producing 6-chlorohydroxyquinone, however TftD can only perform oxidative dechlorination producing 2,6-dichloro-p-quinone. Therefore, a comparative investigation of TftD and TcpA will provide a clear understanding of TcpA’s extra function, indicating how to broaden the substrate range and improve the efficiency of the enzymes. We are determining the 3D-structures followed by site-directed mutagenesis, kinetic and thermodynamic characterization to investigate the unique activities and substrate-specificities.

EmoA is the monooxygenase that oxidizes metal-EDTA complexes, which is the target of our research. NmoA is another monooxygenase catabolizing nitrilotriacetate (NTA), a structural homolog of EDTA and another ubiquitous chelating agent found in everything from agrochemicals to medicines. On the contrary to the recalcitrant character of EDTA in the environment, NTA can be relatively easily degraded by many microorganisms. EmoA and NmoA share a high degree of sequence similarity (36% identity). We found that both NmoA and EmoA can oxidize NTA, but only EmoA can also oxidize EDTA. The mechanistic characterization including 3-D structural information of the two monooxygenases will reveal the critical amino acid residues responsible for the distinct differences. Our efforts could expand the substrate range and the catalytic efficiency of NmoA, which resides in more commonly occurring bacteria. Even though the activity of EmoA resides in their cytoplasm, EDTA-degrading bacteria discovered so far do not degrade stable metal-EDTA complexes. EDTA-degrading bacterium BNC1 has an ABC-type transporter that uptakes free EDTA but not metal-EDTA complexes. The specificity is conferred by the periplasmic binding protein, EppA. Therefore, it is critical to gain mechanistic understanding of the EppA specificity.

In addition, the mechanistic aspects of the flavin reductases (EmoB and TftC) that supply the above monooxygeases with reduced flavins as co-substrates are still unclear. The systematic structural and biophysical approaches to the study of these enzymes will offer information not only about their unique reaction mechanisms but also about the transfer mechanism of reduced flavins.

Lignins, lignans and isoflavonoids are major constituents of vascular plants and account for nearly 30% of the organic carbon circulating in the biosphere. They play important roles in plant defense, helping hosts discriminate between biological pathogens and symbiotic non-pathogens. This research project focuses on determining the structures of various key proteins and enzymes involved in lignan and isoflavonoid biosynthesis, namely the so-called dirigent protein and three reductases: pinoresinol-lariciresinol reductase, phenylcoumaran benzylic ether reductase and isoflavonoid reductase. In addition to their physiological roles in plants, these compounds have pronounced antimicrobial, antifungal, antiviral, antioxidant and anticancer properties. Certain isoflavonoids and lignans are used in medicinal applications, especially for the clinical treatment of various cancers and in cancer prevention. It is generally accepted that low cancer incidence rates in people with specific plant-based diets is due to the so-called chemopreventive effects of particular lignans and isoflavonoids. For these reasons, there is considerable interest in making these compounds more generally available by defining and exploiting the lignan and isoflavonoid biosynthetic pathways. These approaches are seriously hampered, however, by a lack of detailed information on the enzymes involved and the limited success in obtaining them in significant amount. The goal of this project is thus to define the structure-function relationships of these key proteins and enzymes in lignan and isoflavonoid biosynthesis through high-resolution X-ray structural analyses. The information gained from these studies will be very useful for exploring the regulation of lignan and isoflavonoid biosynthesis for ecological plant protection, and for the industrial-scale regiospecific and stereospecific synthesis of these pharmacologically active substances.

Compared to their mesophilic counterparts, thermophilic enzymes have a few kcal/mol additional stabilization energy. The origins of this thermostability should lie within interactions between the constituent amino acid residues, plus cofactor and solvent. In principle, a thorough analysis of the numbers and types of interactions between these constituents may suggest the elements contributing to stabilization and the knowledges learned through such experiments could be used directly in the design of thermostable enzymes. Unfortunately, despite the recent isolation and structural studies of several thermophilic/mesophilic protein pairs, the general structural features or the molecular determinants of protein thermostability that account for the higher thermal stability of thermophilic proteins has not been fully identified.

Here we are investigating structural features that could lead to stabilization of the thermophilic over the mesophilic enzymes through a systematic approach. To study the factors leading to stabilization, we are comparing the detailed structure and thermostability of a number of systematically chosen proteins from a mesophilic and a thermophilic organism.

Electron transfer reactions are the most essential and the simplest processes for life. Not only do they provide the means for transforming solar energy and chemical energy into an utilizable form for all living organisms, they also extend into a range of metabolic processes that support the life of the cell. In life, redox proteins are necessary to control these rates. Differences in “E” of up to a few hundred mV are seen between homologous redox proteins with the same redox site. The site-directed mutagenesis of a single residue can drastically modify this electric potential and subsequently the electron transfer rate. The basic questions, then, are: what are the physical factors that determine the rate constants for protein electron transfer? How do protein structure, and the structure of the redox center itself, influence these factors? To understand this, electron transfer proteins must be studied at an atomic level. We have made systematic mutations at various sites in rubredoxin and ferridoxin to see how these changes influence the redox potential and the local structures. The systematic studies of the crystal structures and the measurement of redox potential could form an essential bridge for electron transfer proteins between their general and the particular behaviors.