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
). The TPDO
KF1
exhibited the substrate specificity for TPA (
k
cat
/
K
m
= 57 ± 9 mM
−1
s
−1
). The TPDO
KF1
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 (
k
cat
) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO.
Importance
The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have 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.
