Molecular insights into substrate recognition and catalysis by phthalate dioxygenase from Comamonas testosteroni

Abstract

Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDO KF1 from Comamonas testosteroni KF1 and found that it had an apparent k cat / K m for phthalate of 0.58 ± 0.09 μM −1 s −1 , over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 Å resolution revealed that it is a hexamer comprising two stacked α 3 trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDO KF1 larger than that of other characterized ROs. Complexes of PDO KF1 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, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.

Biochemical and structural characterization of an aromatic ring–hydroxylating dioxygenase for terephthalic acid catabolism

“…It is possible that iso -PDO and ortho -PDOs from related strains have similar architecture. The strain E6 iso- / ortho -PDO α-subunits share 32% identity with one another, forming a separate lineage from the α-subunits of TPADO and other α 3 β 3 -ROs with which they share ≤19% identity ( 56 ). Accordingly, the α-subunits of TPADO and a recently reported ortho -PDO structure superimpose poorly ( C. testosteroni KF1 PDO) ( SI Appendix , Fig.…”

Engineering Rieske oxygenase activity one piece at a time

“…Based upon approximately 20 unique crystal structures, it has been revealed that Rieske oxygenases assemble into heterohexameric (α 3 β 3 ) [ 21 – 30 ], homohexameric (α 3 α 3 ) [ 31 , 32 ], and homotrimeric (α 3 ) [ 33 – 40 ] architectures ( Figure 2a – c ). Some reports have also suggested that the Rieske oxygenases chlorophyll(ide) a oxygenase and phthalate dioxygenase (PDO) from Micromonas pusilla and Pseudomonas cepacia, respectively, adopt alternative dimeric and tetrameric quaternary structures [ 32 , 41 , 42 ]. However, more recent structural and biochemical data suggest that these systems instead fold into one of the above-noted traditional assemblies [ 32 , 41 , 42 ].…”

“…Some reports have also suggested that the Rieske oxygenases chlorophyll(ide) a oxygenase and phthalate dioxygenase (PDO) from Micromonas pusilla and Pseudomonas cepacia, respectively, adopt alternative dimeric and tetrameric quaternary structures [ 32 , 41 , 42 ]. However, more recent structural and biochemical data suggest that these systems instead fold into one of the above-noted traditional assemblies [ 32 , 41 , 42 ]. In the α 3 β 3 , α 3 α 3 , and α 3 structures, three α subunits, which each contain both metallocenters, arrange into a trimeric architecture that places Rieske cluster and iron site within electron transfer distance of one another across a subunit–subunit interface ( Figure 2a – c ).…”

Identification of New Dioxygenases Able to Recognize Polycyclic Aromatic Hydrocarbons with High Aromaticity

“…PAH degradation by bacteria proceeds through dioxygenase attacks on a PAH to form a cis-dihydrodiol aromatic compound using two atoms of O 2 (Figure 1) [16,17] These proteins are also able to perform oxidation with a high degree of stereo-, regio-, and enantiospecificity at the catalytic mononuclear iron site [18]. Dioxygenases are metalloenzymes present in a wide variety of microorganisms that are able to oxidize PAHs using oxygen molecules and two electrons from a reductase to give cis-arene diols (Figure 1) [19,20]. A wide range of 3D dioxygenase structures have been determined, giving insight into substrate recognition and regioselectivity [19,21].…”