title: Haloalkylphosphorus hydrolases purified from Sphingomonas sp. strain TDK1 and Sphingobium sp. strain TCM1.
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.
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan katsuabe@vos.nagaokaut.ac.jp.
authors.forename: Katsumasa
authors.initials: K
authors.lastname: Abe
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yoshida
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Yuto
authors.initials: Y
authors.lastname: Suzuki
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Junichi
authors.initials: J
authors.lastname: Mori
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Doi
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Shouji
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kera
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003067:Coenzymes / Q000032:analysis; D006842:Hydrocarbons, Brominated / Q000378:metabolism; D006843:Hydrocarbons, Chlorinated / Q000378:metabolism; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010755:Organophosphates / Q000378:metabolism; D044345:Phosphoric Triester Hydrolases / Q000737:chemistry / Q000302:isolation & purification*; D011487:Protein Conformation; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D042301:Sphingomonadaceae / Q000201:enzymology*; D013379:Substrate Specificity; D015032:Zinc / Q000032:analysis
chemical_list: D003067:Coenzymes; D006842:Hydrocarbons, Brominated; D006843:Hydrocarbons, Chlorinated; D010755:Organophosphates; D044345:Phosphoric Triester Hydrolases; D015032:Zinc