STUDIES ON ENZYMES INVOLVED IN PLASTICIZERS DEGRADATION

Abstract

Phthalates are endocrine-disrupting pollutants and potential carcinogens that are extensively used as a plasticizer in a wide range of consumer products, including plastics, medicines, and cosmetics. As phthalates are not covalently bound in these materials, these readily leach into the environment, potentially exposing humans and other organisms to its detrimental health effects through inhalation, ingestion, and absorption. Phthalates are also a major catabolic intermediate in the biodegradation of phthalate esters and widely used plastic polyethylene terephthalate. A wide range of bacterial strains is able to degrade phthalate, isophthalate, and terephthalate. In these strains, the first step in phthalate catabolism is dihydroxylation of the aromatic diacid catalyzed by the phthalate dioxygenase (PDO), terephthalate dioxygenase (TPDO), and isophthalate dioxygenase (IPDO). All these enzymes are Rieske oxygenases (ROs). ROs are a family of enzymes best known for catalyzing the NAD(P)H-dependent dihydroxylation of aromatic compounds pollutants. RO systems comprise two or three components: an oxygenase and a reductase that transfers electrons from NAD(P)H to the oxygenase, either directly or via a ferredoxin. The oxygenase contains a mononuclear iron where catalysis occurs and a Rieske-type iron-sulfur cluster ([2Fe-2S]) that mediates electron transfer to the catalytic center. Structural studies on ROs such as naphthalene dioxygenase (NDO) have identified key features that are responsible for substrate specificity and have provided valuable insight into the catalytic mechanism of these enzymes. With regard to phthalate degradation, the PDO systems comprise two components: an oxygenase and a reductase, encoded by phtA and phtB, respectively. Work on PDO from Burkholderia cepacia DB01 (PDODB01) pioneered our understanding of RO function. However, despite extensive efforts, the structure of PDO has been elusive. Moreover, PDOs share less than 20% amino acid sequence identity with other structurally characterized ROs. Thus, it has been difficult to identify key residues beyond the metal ligands and an acidic residue proposed to mediate the interaction between the Rieske cluster and the mononuclear iron. With regard to terephthalate degradation, previous studies have established that the TPDO enzyme system comprises two components: an oxygenase and a reductase, terephthalate dioxygenase reductase (TPDR), encoded by tphA2A3 and tphA1, respectively. TPDO is the catalytic component of the enzyme system and is made up of α and β subunits. The α subunit contains Rieske [2Fe-2S] center and a mononuclear iron center for catalysis.Despite these pieces of information, the identification of crucial residues governing the TPA dihydroxylation has been elusive due to the lack of crystal structure of TPDO. Similarly, with regard to isophthalate degradation, previous studies have identified and characterized IPDO enzyme system. However, it has not been studied concerning its structural aspects. Thus, the functional residues of this enzyme are not known. In this context, biochemical and structural characterization of PDO from Comamonas testosteroni KF1 (PDOKF1), IPDO from C. testosteroni KF1 (IPDOKF1), TPDO from C. testosteroni KF1 (TPDOKF1), and a phthalate binding protein, PhtE, which helps in transporting phthalate into the bacterial cell was performed. The first chapter discusses the literature related to phthalates, microbial degradation of phthalates, microbial phthalate degradation pathways, RO enzymes, and solute transport systems. It briefs about different types of RO family enzymes catalyzing the first step in the oxidative degradation of aromatic and different kinds of transport systems for cellular uptake of solute molecules. Finally, the importance of the structural characterization of these enzymes is discussed. The second chapter describes the characterization of PDOKF1 and a Rieske Oxygenase (ROCH34) from Cupriavidus metallidurans CH34. Firstly, genes encoding these enzymes were cloned, expressed, and heterologously purified from E. coli BL21 (DE3). Biochemical characterization of these enzymes was performed with HPLC and Oxygraph assay. These enzymes were crystallized and structures were determined with SAD Phasing and Molecular Replacement techniques. With crystal structure, we show that the PDOKF1 and ROCH34 protomers adopt a head-to-tail configuration typical of ROs. Complexes of PDOKF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, the substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. The third chapter describes the biochemical and structural characterization of TPDOKF1. We characterized the steady-state kinetics and first crystal structure of TPDOKF1. The TPDOKF1 exhibited the substrate specificity for TPA (kcat/Km = 57 ± 9 mM−1s−1). The TPDOKF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity (kcat) by 80%.Altogether, our biochemical and structural information delineates the structural features that dictate the substrate recognition and specificity of TPDOKF1. The next chapter describes the structural characterization of isophthalate dioxygenase (IPDO) from C. testosteroni KF1. The gene encoding this enzyme was cloned, expressed, and heterologously purified from E. coli BL21 (DE3). The IPDO enzyme was crystallized and structure was determined with the Molecular Replacement technique. The crystal structure shows typical trimeric architecture and head-to-tail arrangement of Rieske Oxygenases. The isophthalate binding mode was determined with in-silico molecular docking tools and key residues governing the isophthalate recognition are identified to be Arg234 and His257. The fifth chapter describes the characterization of a phthalate binding protein, PhtE from C. testosteroni KF1. The PhtE protein was crystallized in apo form as well as in complex with phthalate and structures were determined with the Molecular Replacement technique. The phthalate binding mode was analyzed with the crystal structure of the PhtE-phthalate complex and key residues conferring the phthalate interaction in the solute binding cavity are identified. The residues His167, Ser190, and Ser208 are observed to be crucial for phthalate binding, while residues Tyr40, Leu45, Pro46, and Asp96 entrap the phthalate molecule inside the substrate-binding cleft. The sixth chapter, concludes the studies performed in this thesis towards characterizing structural and biochemical features of four RO enzymes and a transporter. Finally, the scope and the future perspectives are discussed.