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Haloalkylphosphorus hydrolases purified from Sphingomonas sp. strain TDK1 and Sphingobium sp. strain TCM1.

Applied and environmental microbiology (2014-07-20)
Katsumasa Abe, Satoshi Yoshida, Yuto Suzuki, Junichi Mori, Yuka Doi, Shouji Takahashi, Yoshio Kera
ABSTRACT

Phosphotriesterases catalyze the first step of organophosphorus triester degradation. The bacterial phosphotriesterases purified and characterized to date hydrolyze mainly aryl dialkyl phosphates, such as parathion, paraoxon, and chlorpyrifos. In this study, we purified and cloned two novel phosphotriesterases from Sphingomonas sp. strain TDK1 and Sphingobium sp. strain TCM1 that hydrolyze tri(haloalkyl)phosphates, and we named these enzymes haloalkylphosphorus hydrolases (TDK-HAD and TCM-HAD, respectively). Both HADs are monomeric proteins with molecular masses of 59.6 (TDK-HAD) and 58.4 kDa (TCM-HAD). The enzyme activities were affected by the addition of divalent cations, and inductively coupled plasma mass spectrometry analysis suggested that zinc is a native cofactor for HADs. These enzymes hydrolyzed not only chlorinated organophosphates but also a brominated organophosphate [tris(2,3-dibromopropyl) phosphate], as well as triaryl phosphates (tricresyl and triphenyl phosphates). Paraoxon-methyl and paraoxon were efficiently degraded by TCM-HAD, whereas TDK-HAD showed weak activity toward these substrates. Dichlorvos was degraded only by TCM-HAD. The enzymes displayed weak or no activity against trialkyl phosphates and organophosphorothioates. The TCM-HAD and TDK-HAD genes were cloned and found to encode proteins of 583 and 574 amino acid residues, respectively. The primary structures of TCM-HAD and TDK-HAD were very similar, and the enzymes also shared sequence similarity with fenitrothion hydrolase (FedA) of Burkholderia sp. strain NF100 and organophosphorus hydrolase (OphB) of Burkholderia sp. strain JBA3. However, the substrate specificities and quaternary structures of the HADs were largely different from those of FedA and OphB. These results show that HADs from sphingomonads are novel members of the bacterial phosphotriesterase family.

MATERIALS
Product Number
Brand
Product Description

Supelco
Chlorpyrifos, certified reference material, TraceCERT®
Supelco
Chlorpyrifos, PESTANAL®, analytical standard
Supelco
Parathion, PESTANAL®, analytical standard
Supelco
Dichlorvos, PESTANAL®, analytical standard
Supelco
TDCPP, PESTANAL®, analytical standard
Sigma-Aldrich
Triphenyl phosphate solution, NMR reference standard, 0.0485 M in acetone-d6 (99.9 atom % D)
Supelco
Tributyl phosphate, Pharmaceutical Secondary Standard; Certified Reference Material
Tri-n-butyl phosphate, European Pharmacopoeia (EP) Reference Standard
Supelco
Triphenyl phosphate solution, NMR reference standard, 3 mM in chloroform-d (99.8 atom % D), NMR tube size 5 mm × 8 in.
Supelco
Triphenyl phosphate solution, NMR reference standard, 0.0485 M in acetone-d6 (99.9 atom % D), NMR tube size 5 mm × 8 in.
Sigma-Aldrich
Triphenyl phosphate solution, NMR reference standard, 0.0485 M in acetone-d6 (99.9 atom % D), NMR tube size 10 mm × 8 in.
Sigma-Aldrich
Triphenyl phosphate solution, NMR reference standard, 0.0485 M in acetone-d6 (99.9 atom % D), NMR tube size 3 mm × 8 in.
Sigma-Aldrich
Tributyl phosphate, ≥99%
Supelco
Triphenyl phosphate solution, certified reference material, 500 μg/mL in methyl tert-butyl ether
Supelco
Triphenyl phosphate, analytical standard
Supelco
Triphenyl phosphate solution, NMR reference standard, 0.0485 M in chloroform-d (99.8 atom % D), NMR tube size 5 mm × 8 in.
Sigma-Aldrich
Tributyl phosphate, 97%
Sigma-Aldrich
Triphenyl phosphate, ≥99%
Sigma-Aldrich
Triethyl phosphate, ReagentPlus®, ≥99.8%
Sigma-Aldrich
Trimethyl phosphate, 97%
Supelco
Tributyl phosphate, for extraction analysis, ≥99.0% (GC)
Sigma-Aldrich
Trimethyl phosphate, ≥99%
Supelco
Paraoxon-ethyl, PESTANAL®, analytical standard
Sigma-Aldrich
Tributyl phosphate, puriss., ≥99.0% (GC)
Supelco
Triphenyl phosphate solution, NMR reference standard, 0.0485 M in chloroform-d (99.8 atom % D), NMR tube size 10 mm × 8 in.
Sigma-Aldrich
Paraoxon-ethyl, ≥90%, oil