Structure of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa at 2.15 Å resolution

Protocatechuate 3,4-dioxygenase catalyzes the aromatic ring cleavage of 3,4-dihydroxybenzoate by incorporating both atoms of molecular oxygen to yield β-carboxy-cis, cis-muconate. The structure of this metalloenzyme from Paeudomonas aeruginosa (now reclassified as P. putida) has been refined to an R-factor of 0.172 to 2.15 Å resolution. The structure is a highly symmetric (αβFe³⁺)₁₂ aggregate with a root-mean-square (r.m.s.) difference of 0.18 Å among symmetry-related atoms. The tertiary structure of the two polypeptides (α and β) are highly homologous (r.m.s. difference of 1.05 Å over 127 C^I atoms), suggesting that the ancestral enzyme was originally a homodimer with two active sites. Indeed, a non-functional, vestigial active site retains many of the properties of the functional active site but does not bind iron. The coordination geometry of the non-heme iron catalytic cofactor can best be described as trigonal bipyramidal with Tyr447 (147β) and His462 (162β) serving as axial ligands, and Tyr408 (108β), His460 (160β) and Wat837 serving as equitorial ligands. The active site environment has a number of basic residues that may promote binding of the acidic substrate. Within the putative active site cavity which is located between α and β chains, five approximately coplanar solvent molecules suggest a position for the planar substrate Trp449 (149β), Ile491 (191β), defined by Gly14 (14α) and Pro15 (15α). In this position the guanidino group of Arg457 (157β) would be buried by the substrate, suggesting a functional role in catalysis.