7V25

Crystal Structure of phthalate dioxygenase in complex with phthalate

Summary for 7V25

• Entry DOI: 10.2210/pdb7v25/pdb
• Descriptor: Rieske (2Fe-2S) domain protein, FE (II) ION, FE2/S2 (INORGANIC) CLUSTER, ... (5 entities in total)
• Functional Keywords: dioxygenase, bacterial protein, metalloprotein, rieske domain, oxidoreductase
• Biological source: Comamonas testosteroni (strain DSM 14576 / KF-1)
• Total number of polymer chains: 6
• Total formula weight: 296566.42

Authors

Mahto, J.K.,Kumar, P. (deposition date: 2021-08-07, release date: 2021-12-15, Last modification date: 2023-11-29)

Primary citation

Mahto, J.K.,Neetu, N.,Waghmode, B.,Kuatsjah, E.,Sharma, M.,Sircar, D.,Sharma, A.K.,Tomar, S.,Eltis, L.D.,Kumar, P.
Molecular insights into substrate recognition and catalysis by phthalate dioxygenase from Comamonas testosteroni.
J.Biol.Chem., 297:101416-101416, 2021

PubMed 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 from Comamonas testosteroni KF1 and found that it had an apparent k/K for phthalate of 0.58 ± 0.09 μMs, 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 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 larger than that of other characterized ROs. Complexes of PDO 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.

PubMed: 34800435
DOI: 10.1016/j.jbc.2021.101416

Experimental method

X-RAY DIFFRACTION (2.74 Å)