BIOCHEMICAL AND STRUCTURAL STUDIES ON DEHYDROGENASES OF PHTHALATE AND ISOPHTHALATE DEGRADATION PATHWAYS

Abstract

Phthalates are endocrine disruptors with carcinogenic potential widely utilized as plasticizers in various consumer items such as plastics, medications, and cosmetics. As phthalates are not covalently bonded in these materials, they easily leak into the environment, possibly exposing people and other species to harmful health consequences via inhalation, ingestion, and absorption. Proteobacteria such as Burkholderia cepacia DB01 and Comamonas testosteroni and actinobacteria such as Rhodococcus jostii RHA1 and Arthrobacter keyseri 12B can thrive on plasticizers. The initial stage in phthalate catabolism in these strains is dihydroxylation of the aromatic diacid, mediated by Rieske oxygenases such as phthalate dioxygenase and isophthalate dioxygenase that produce cis-diol products. The second step of the degradation process involves the dehydrogenation of the re-aromatization of the cis-diol phthalate. In the phthalate catabolic pathway, based on the position of the ring-hydroxylation, phthalate dioxygenase is either phthalate 4,5-dioxygenase, which is found mainly in Gramnegative bacteria, or phthalate 3,4-dioxygenase, which is found primarily in Gram-positive bacteria. The action of phthalate 4,5-dioxygenase results in cis-4,5-dihydroxy-4,5- dihydrophthalate, which is dehydrogenated to 4,5-dihydroxyphthalate by cis-phthalate dihydrodiol dehydrogenase. This dihydroxylated phthalate is decarboxylated by 4,5- dihydroxyphthalate decarboxylase to give 3,4-dihydroxybenzoate. The action of phthalate 3,4-dioxygenase results in the formation of cis-3,4-dihydroxy-3,4-dihydrophthalate, which is then transformed to 3,4-dihydroxyphthalate and decarboxylated to create 3,4- dihydroxybenzoate by 3,4-dihydroxyphthalate decarboxylase. The phthalate cis-dihydrodiol dehydrogenase (PhtC) is one of the crucial enzymes of the phthalate catabolic pathway. The PhtC is an oxidoreductase belonging to the Gfo/Idh/MocA protein family with EC niche (EC 1.3.1.64). There are other cis-dihydrodiol dehydrogenases that have been studied with respect to the biodegradation of terephthalate, chlorobenzene, 3-chlorobenzoate, naphthalene, isophthalate, and biphenyl. As compared to the cis-dihydrodiol dehydrogenases that belong to the short-chain dehydrogenase family (SDRs), very little information is known about the long-chain cis-dihydrodiol dehydrogenases. Moreover, he cis-dihydrodiol dehydrogenases family enzymes have remained underexplored because of the lack of structural information on its representatives, with only a few structures available in the PDB database, which warrants further structural studies of this physiologically important group of enzymes. Prior works have identified the genes responsible for the isophthalate catabolism, and the disruption of these genes conferred the microbes an inability to thrive on isophthalate. According to a metabolomic analysis, isophthalate dioxygenase doubly hydroxylates it to form 1,2-dihydroxy-3,5-cyclohexadiene-1,5-dicarboxylate (1,5-DCD), which is subsequently decarboxylated to 3,4-dihydroxybenzoate by 1,2-dihydroxy-3,5-cyclohexadiene-1,5- dicarboxylate (1,5-DCD). Several decarboxylating NAD(P)+-dependent dehydrogenases, such as 2-hydro-cis-1,2-dihydroxy-terephthalate dehydrogenases and 1,2-dihydroxy-3,5- cyclohexadiene-1,5-dicarboxylate dehydrogenase have been identified. In this case, dehydrogenation occurs by removing a hydride from one carbon and then decarboxylating the neighboring carbon. The structural characterization of PhtCKF1 and IphBKF1 was carried out in this context. The apparent specificities of the enzymes were evaluated using steady-state kinetics. The crystal structures for apo-enzyme, cofactor-bound binary complex and ternary complex of the enzyme with cofactor and product analog were determined. With site-directed mutagenesis, the role of the residues in substrate positioning was investigated. Chapter 1 reviews the literature related to plastic pollution and describes the various ways to degrade plastic. Despite applying various physical and chemical methods involved in plastic and plasticizers degradation, bioremediation stands out as the most economical and eco-friendly way of completely degrading these xenobiotics. This chapter focuses on the various microbes and the various pathways involved in the bio-degradation of such xenobiotics. The crucial role of various enzymes involved in the catabolic pathways is described. Chapter 2 describes the characterization of phthalate cis-dihydrodiol dehydrogenase from Comamonas testosteroni KF1 (PhtCKF1). The gene encoding the enzyme was cloned, expressed, and heterologously purified from E. coli BL21 (DE3). Biochemical characterization of these enzymes was performed. These enzymes were crystallized, and the structure was determined with Molecular Replacement. The binary and ternary structures with NAD+ and 3-hydroxy benzoate were determined. The comparison of these structures revealed the conformational flexibility of the binding loop. With site-directed mutagenesis, the roles of critical residues in substrate specificity and catalysis were evaluated. Chapter 3 describes the characterization of isophthalate dehydrogenase from Comamonas testosteroni KF1. The gene encoding this enzyme was cloned, expressed, and heterologously purified from E. coli BL21 (DE3). Biochemical characterization of this enzyme was performed. The dehydrogenation activity was assessed through steady-state kinetics. This enzyme was crystallized, and its structure was determined with the Molecular Replacement technique. The binary structure of the protein was determined in a complex with NADP+. The binding mode of the substrate was predicted, and critical residues governing the substrate binding were revealed. The comparison of the binary and apo structures revealed the significance of the conformational flexibility of the substrate binding loop. Chapter 4 concludes the work done in this thesis in which biochemical and biophysical analyses showed distinct structural and biochemical properties of the two cis-diol dehydrogenases. The essential residues determining each enzyme's substrate-specificity and regio-specificity were identified. The scope and future prospects are examined.