Sphingobium Chlorophenolicum Dichlorohydroquinone Dioxygenase (PcpA) Is Alkaline Resistant and Thermally Stable

Abstract

Dichlorohydroquinone dioxygenase (PcpA) is the ring-cleavage enzyme in the PCP biodegradation pathway in Sphingobium chlorophenolicum strain ATCC 39723. PcpA dehalogenates and oxidizes 2,6-dichlorohydroquinone to form 2-chloromaleylacetate, which is subsequently converted to succinyl coenzyme A and acetyl coenzyme A via 3-oxoadipate. Previous studies have shown that PcpA is highly substrate-specific and only uses 2,6-dichlorohydroquinone as its substrate. In the current study, we overexpressed and purified recombinant PcpA and showed that PcpA was highly alkaline resistant and thermally stable. PcpA exhibited two activity peaks at pH 7.0 and 10.0, respectively. The apparent kcat and Km were measured as 0.19 ± 0.01 s-1 and 0.24 ± 0.08 mM, respectively at pH 7.0, and 0.17 ± 0.01 s-1 and 0.77 ± 0.29 mM, respectively at pH 10.0. Electron paramagnetic resonance studies showed rapid oxidation of Fe(II) to Fe(III) in PcpA and the formation of a stable radical intermediate during the enzyme catalysis. The stable radical was predicted to be an epoxide type dichloro radical with the unpaired electron density localized on C3.

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

“…Purified PcpA lost activity in a time‐dependent manner (Supplementary Fig. S1) by autoxidation of Fe(II) to Fe(III) (Xun et al ., ; Sun et al ., ), which could be completely recovered in 48 h by reduction with 100 μM ascorbic acid. PcpA recovered from crystals that were dissolved in solution were found to be catalytically inactive, but regained full activity after reduction by ascorbic acid (Supplementary Fig.…”

“…PcpA uses Fe(II) for catalysis (Xun et al ., ; Sun et al ., ) which is partially oxidized to Fe(III) during purification (Xun et al ., ; Sun et al ., ). Purified PcpA lost activity in a time‐dependent manner (Supplementary Fig.…”

Identification and Characterization of Two Endogenous β-Glucosidases from the Termite Coptotermes formosanus

“…The observation of two peaks in pH profile was confirmed by repeating the experiment multiple times under finer grids around pH 8.0. This double-peak peaks [31]. However, it has been reported that BGs from the termite N. takasagoensis, N. koshunensis, and C. formosanus are most active at pH 5.0-5.5 and inactivated at extreme pH (<3.0 or >8.0) [15,19,29,32].…”

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).…”