Department of Biosciences and Bioengineering, IIT Roorkee, Roorkee, Uttarakhand, India.
Department of Civil Engineering, IIT Roorkee, Roorkee, Uttarakhand, India.
J Bacteriol. 2022 Mar 15;204(3):e0054321. doi: 10.1128/JB.00543-21. Epub 2022 Jan 10.
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 -diol, is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO). TPDO exhibited substrate specificity for TPA (/ = 57 ± 9 mM s). The TPDO 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 () by 80%. This study delineates the structural features that dictate the substrate recognition and specificity of TPDO. 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.
邻苯二甲酸酯（TPA）的生物降解是细菌利用这种聚对苯二甲酸乙二醇酯（PET）解聚产物的高度期望的代谢过程，但迄今为止，邻苯二甲酸二加氧酶（TPDO）的结构仍然未知，TPDO 是一种 Rieske 氧合酶（RO），可催化 TPA 双羟化生成 -二醇。在这项研究中，我们对来自康宁氏杆菌 KF1 的邻苯二甲酸二加氧酶（TPDO）进行了稳态动力学和首次晶体结构的表征。TPDO 对 TPA 表现出底物特异性（/ = 57 ± 9 mM s）。TPDO 结构具有特征性的 RO 特征以及独特的催化结构域，合理化了酶的功能。对接和突变研究表明，其对 TPA 的底物特异性是由位于活性位点相对面上的 Arg309 和 Arg390 残基介导的。此外，残基 Gln300 也被证明对活性至关重要，因为将其突变为丙氨酸会使活性降低 80%（）。这项研究阐明了决定 TPDO 底物识别和特异性的结构特征。
全球塑料污染已成为最紧迫的环境问题。最近关于酶将聚乙烯 terephthalate 塑料解聚成 terephthalate (TPA) 的研究显示出解决这个问题的一些潜力。微生物利用这种释放的产物 TPA 是一种新兴的、有前途的废物变价值的策略。在过去的十年中，研究已经确定邻苯二甲酸二加氧酶（TPDO）是负责在少数革兰氏阴性和革兰氏阳性细菌中启动 TPA 酶促降解的酶。在这里，我们确定了来自康宁氏杆菌 KF1 的 TPDO 的晶体结构，并揭示了它具有独特的催化结构域，在活性位点具有两个碱性残基，用于识别 TPA。生化和突变研究表明，负责该酶底物特异性的关键残基。
