Structural Insights into Dihydroxylation of Terephthalate, a Product of Polyethylene Terephthalate Degradation

Abstract

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis-diol, is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO_KF1). TPDO_KF1 exhibited substrate specificity for TPA (k_cat/K_m = 57 ± 9 mM^-1 s^-1). The TPDO_KF1 structure harbors characteristic 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 for TPA is mediated by the Arg309 and Arg390 residues, positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, as its mutation to alanine decreases the activity (k_cat) by 80%. This study delineates the structural features that dictate the substrate recognition and specificity of TPDO. IMPORTANCE Global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential for tackling this. Microbial utilization of this released product, TPA, is an emerging and promising strategy for waste-to-value creation. Research in the last decade has identified terephthalate dioxygenase (TPDO) as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

Keywords: Comamonas testosteroni KF1; Rieske center; mononuclear iron; polyethylene terephthalate; terephthalate dioxygenase.

FIG 1

Terephthalate catabolic pathway, biochemical characterization, steady-state kinetics, and phylogenetic tree of TPDO_KF1. (A) Schematic for the terephthalate catabolic pathway that ends with the formation of protocatechuate, which is eventually metabolized into TCA cycle intermediates. The first reaction of the pathway is catalyzed by the terephthalate-1,2-dioxygenase system. (B) Phylogenetic relationship of TPDO_KF1. The unrooted phylogenetic tree was calculated using a sequence alignment of 18 Rieske oxygenase α subunits. The proteins are abbreviated using the protein name and strain as follows (GenPeptID): biphenyl dioxygenase from Comamonas testosteroni B356, BPDO_B356 (AAC44526); Paraburkholderia xenovorans LB400, BPDO_LB400 (AAB63425); Rhodococcus jostii RHA1, BPDO_RHA1 (BAA06868); naphthalene dioxygenase from Pseudomonas putida 9816-4, NDO_Pp (P0A110); Rhodococcus sp. strain NCIMB 12038, NDO_Rho (AAD28100); 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2, NTDO_DS2 (AGH09226); nitrobenzene dioxygenase from Comamonas sp. strain JS765, NBDO_JS765 (AAL76202); salicylate hydroxylase from Ralstonia sp. strain U2, NagGH (AAD12607); anthranilate dioxygenase from Burkholderia cepacia DB01, AntDO_DB01 (Q84BZ3); terephthalate dioxygenase from Comamonas testosteroni E6, TPDO_E6 (AIJ48578); R. jostii RHA1, TPDO_RHA1 (ABH00392); benzoate dioxygenase from Pseudomonas putida, BODO_Pp (WP_011600771); carbazole dioxygenase from Sphingomonas sp. strain GTIN11, CarDO_GTIN11 (AAL37976); dicamba monooxygenase from Stenotrophomonas maltophilia, DMO_Sma (AAV53699); phthalate dioxygenase from Rhodococcus jostii RHA1, PDO_RHA1 (BAD36800); Burkholderia cepacia DB01, PDO_DB01 (WP_011881604); 3-chlorobenzoate dioxygenase from Comamonas testosteroni BR60, CbaDO (Q44256); isophthalate dioxygenase from Comamonas testosteroni E6, IPDO_E6 (BAH70269). (C) SDS-PAGE analysis of TPDO_KF1 (L1) and TPDR_KF1 (L3) purified fractions using Ni-NTA chromatography, depicting α subunit (∼49 kDa), β subunit (∼17 kDa), and TPDR_KF1 (∼38 kDa). (D) Elution profiles of standard proteins obtained by size exclusion chromatography using a Superdex200 column. The molecular masses of the standard proteins are as follows: ferritin, 440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa; and ovalbumin, 44 kDa. (Inset) Analytical curve based on the elution volumes of standard proteins from the chromatographic results. The chromatogram shows a predominant peak at 11 mL, corresponding to the molecular mass of the TPDO_KF1 heterohexamer (∼210 kDa), and minor peaks at 8.5 and 13 mL, corresponding to the molecular mass of TPDO_KF1 as an oligomer equivalent to 4(α₃β₃) and as an αβ heterodimer, respectively. (E) Conversion of terephthalate by purified TPDO_KF1 and TPDR_KF1. Purified enzymes (10 μM each) were incubated with 500 μM terephthalate in the presence of 100 μM NAD(P)H. (F) MS m/z values of the putative product formed at retention time 2.24 min; the m/z value of 200.03 represents the mass of dihydroxylated terephthalic acid. (G) Dependence of initial velocity of oxygen consumption (V₀, in μM min⁻¹) on the terephthalate concentration (μM) in the air-saturated buffer. The red line represents the fitting of the Michaelis-Menten equation to the data.

FIG 2

Amino acid sequence alignment of the α subunit of TPDO_KF1 with the α subunits of related ROs. The aligned sequences are TPDO from Comamonas testosteroni KF1 (TPDO-KF1), TPDO from Rhodococcus jostii RHA1 (TPDO-RHA1), salicylate hydroxylase from Ralstonia sp. strain U2 (NagGH-U2), biphenyl dioxygenase from Comamonas testosteroni B356 (BPDO-B356), and phthalate dioxygenase from Rhodococcus jostii RHA1 (PDO-RHA1). Identical residues have a black background. Similar residues are highlighted with blue boxes. The purple background highlights residues involved in coordinating Rieske centers and mononuclear iron centers. The red background highlights residues that interact with the substrate. The program ESPript was used for visualization.

FIG 3

Crystal structure of TPDO_KF1. (A) Cartoon representation of TPDO_KF1 α₃β₃ hexamer; subunits A, B, C, D, E, and F are in green, teal, blue, red, salmon, and orange, respectively. (B) Residue Asp207 was found at the subunit-subunit interface bridge from the Rieske center to the catalytic mononuclear iron center. (C) Coordination of the mononuclear iron center with residues and two water molecules. (D and E) Cartoon representations of TPDO_KF1 with secondary structure elements, the α monomer (D) and the β monomer (E).

FIG 4

Structural comparison of TPDO_KF1 with its homolog ROs. (A) The TPDO_KF1 structure is superposed with its closest structural homologs. Cartoon representations of TPDO_KF1 (cyan), NagGH (gray), BPDO_RHA1 (blue), BPDO_LB400 (pink), and BPDO_B356 (yellow) are shown. (B) Structural superposition of the β subunit of TPDO_KF1 with β subunits of related ROs. The shortened αA helix, different spatial position of αD helix, and absence of the extended region between the βA and βB β strands are depicted. (C) The NagGH structure lacks the active-site entrance structural element (circled in black) and the two-stranded antiparallel β sheet (β1 and β18) at the N-terminal region (red box). The α-helices of NagGH are also seen to not align with the TPDO_KF1 structure. (D) With BPDOs, the α-helices of the TPDO_KF1 catalytic domain were observed to not superpose; the regions are highlighted. (E) The active-site entrance region of TPDO_KF1 is different from that of BPDOs, and it constitutes an α helix at the region equivalent to that in BPDOs where it constitutes four α-helices. (F) The solvent-accessible surface representation of the TPDO_KF1 active site shows that there are two cavity openings. (G) The active-site tunnel openings of TPDO_KF1 are blocked with structural elements in BPDO_LB400.; opening 1 and opening 2 are blocked by a loop element and an α helix element of BPDO_LB400, respectively.

FIG 5

Active site of TPDO_KF1. (A) The surface representation of TPDO_KF1 (gray) shows that the two openings of the active-site tunnel are formed by residues 208 to 230 (opening 1) and residues 245 to 287 (opening 2). (B) The TPDO_KF1 active-site cavity can be divided into three subsites: proximal (P), middle (M), and distal (D). Subsites with constituent residues are labeled. (C) The TPA-docked TPDO_KF1 structure reveals crucial residues. The interactions of TPA with TPDO_KF1 active-site residues are highlighted. The interactions are represented with black dotted lines. Residues and bond distances (in Å) are shown. (D) Conformational changes in Arg390 and Gln300 residues, as obtained from flexible docking (cyan) compared to the apo structure (gray).

FIG 6

Active-site comparison of TPDO_KF1 with related ROs. (A) Superposition of active-site residues of TPDO_KF1 (cyan) with NagGH (magenta). The substitution of Ile302 at the corresponding position of Gln316 of NagGH may account for the inability of TPDO_KF1 to oxygenate salicylate. (B) Superposition with BPDO_B356 (yellow) shows that the TPDO_KF1 structure lacks the corresponding aromatic biphenyl ring-interacting residues, especially Phe277, Phe376, and Phe382. (C and D) The superposition of TPDO_KF1 with (C) PDO_KF1 (green) and (D) PDO_RHA1 (violet). The equivalents of Arg207 and Arg218 (phthalate-interacting residues of PDO_KF1 and PDO_RHA1, respectively) are absent in the TPDO_KF1 structure.