Structural characterization of 2,6‐dichloro‐p‐hydroquinone 1,2‐dioxygenase (PcpA) from Sphingobium chlorophenolicum, a new type of aromatic ring‐cleavage enzyme

Abstract

Summary PcpA (2,6-dichloro-p-hydroquinone 1,2-dioxygenase) from Sphingobium chlorophenolicum, a non-haem Fe(II) dioxygenase capable of cleaving the aromatic ring of p-hydroquinone and its substituted variants, is a member of the recently discovered p-hydroquinone 1,2-dioxygenases. Here we report the 2.6 Å structure of PcpA, which consists of four βαβββ motifs, a hallmark of the vicinal oxygen chelate superfamily. The secondary co-ordination sphere of the Fe(II) centre forms an extensive hydrogen-bonding network with three solvent exposed residues, linking the catalytic Fe(II) to solvent. A tight hydrophobic pocket provides p-hydroquinones access to the Fe(II) centre. The p-hydroxyl group is essential for the substrate-binding, thus phenols and catechols, lacking a p-hydroxyl group, do not bind to PcpA. Site-directed mutagenesis and kinetic analysis confirm the critical catalytic role played by the highly conserved His10, Thr13, His226 and Arg259. Based on these results, we propose a general reaction mechanism for p-hydroquinone 1,2-dioxygenases.

Characterizations of Two Bacterial Persulfide Dioxygenases of the Metallo-β-lactamase Superfamily

“…Given the fact that most members of the metallo-␤-lactamase superfamily are hydrolytic enzymes with binuclear metal centers (26), PDOs likely evolved from a hydrolytic enzyme that has two coordinated metal ions. Evolution has led to loss of affinity for a second metal and gain of the coordination for water molecules together with a hydrogen bond network in their second coordination sphere, which is likely critical to Fe(II) positioning and catalysis, as we proposed in the case of 2,6-dichloro-p-hydroquinone 1,2-dioxygenase (27). Consequently, the metalbinding center is highly conserved among PDOs.…”

Hydroquinone: Environmental Pollution, Toxicity, and Microbial Answers

“…PcpA protein contains a nonheme Fe atom and has no homology with classical ring-fission enzymes such as catechol dioxygenase. The enzyme is able to catalyze the ring-opening of a wide range of substituted hydroquinones [60, 63, 64]. In spite of the detailed knowledge of the PcpA enzyme from S. chlorophenolicum , other putative members of the family are present in several gram-negative bacteria (Figure 3).…”

A two-electron-shell game: intermediates of the extradiol-cleaving catechol dioxygenases

“…(A) HPCD, homoprotocatechuate 2,3-dioxygenase [9]; (B) DHBD, 2,3-dihydroxybiphenyl 1,2-dioxygenase [10]; (C) 4,5-PCD, protocatechuate 4,5-dioxygenase [11]; (D) HAD, 3-hydroxyanthranilate-3,4-dioxygenase [2]; (E) HGD, homogentisate 1,2-dioxygenase[12]; (F) PcpA, 2,6-dichloro- p -hydroquinone 1,2-dioxygenase [4]. Four-letter codes adjacent to each reaction represent PDB files for respective enzymes.…”

“…These enzymes are most often found in soil bacteria and serve to transform a range of substrates with 1,2-dihydroxybenzene moieties into ring-cleaved aliphatic products that eventually re-enter the Krebs cycle (Scheme 1A–C) [1]. Mechanistically similar enzymes that utilize substrates in which one of ortho -hydroxyl groups is replaced by an amine (Scheme 1D) [2] or where the hydroxyl groups are para to one another (Scheme 1E&F) have also been studied [3, 4]. Other extradiol cleaving catechol dioxygenases are involved in natural product biosynthesis pathways [5].…”