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.

FIG 1

SDS-polyacrylamide gel electrophoresis of HADs from Sphingomonas sp. strain TDK1 (A) and Sphingobium sp. strain TCM1 (B). Protein samples were separated on a 12.5% SDS-polyacrylamide gel and stained with Coomassie brilliant blue R-250. (A) Lane 1, marker proteins; lane 2, crude extract (10 μg protein); lane 3, ammonium sulfate fractionation (10 μg); lane 4, butyl-Sepharose fraction (2 μg); lane 5, SP-Sepharose fraction (2 μg); lane 6, Sephacryl S-200 fraction (2 μg). (B) Lane 1, marker proteins; lane 2, crude extract (10 μg protein); lane 3, ammonium sulfate fractionation (10 μg); lane 4, dialyzed solution applied to SP-Sepharose (10 μg); lane 5, SP-Sepharose fraction (4 μg); lane 6, phenyl-Sepharose fraction (4 μg).

FIG 2

Effect of pH (A and C) and temperature (B and D) on TDK-HAD (A and B) and TCM-HAD (C and D) activity. (A) Enzyme activity was measured in 50 mM sodium phosphate buffer (closed squares), 50 mM Tris-HCl buffer (closed circles), and 50 mM glycine-NaOH buffer (closed triangles). The enzyme activity was assayed at 30°C for 30 min. (B) Enzyme activity was assayed in 50 mM Tris-HCl buffer (pH 8.0) at 10 to 50°C for 30 min. (C) Enzyme activity was measured in 50 mM sodium phosphate buffer (closed squares), 50 mM morpholineethanesulfonic acid buffer (open triangles), 50 mM piperazine-N,N′-bis(2-ethanesulfonic acid) buffer (open circles), 50 mM Tris-HCl buffer (closed circles), 50 mM N-cyclohexyl-2-aminoethanesulfonic acid buffer (open squares), and 50 mM glycine-NaOH buffer (closed triangles). The enzyme activity was assayed at 25°C for 30 min. (D) Enzyme activity was assayed in 50 mM Tris-HCl buffer (pH 9.0) at 10 to 50°C for 30 min. TDCPP (50 μM) or TCEP (1 mM) was used as the substrate for the assay of TDK-HAD (A and B) and TCM-HAD (C and D), respectively. The final concentrations of purified TDK-HAD and TCM-HAD in these assay mixtures were 0.41 μg/ml and 2.8 μg/ml, respectively.

FIG 3

Effect of substrate concentration on the activity of TCM-HAD. The assay mixture contained different concentrations of TCEP, 50 mM Tris-HCl buffer (pH 9.0), and 2.8 μg/ml of TCM-HAD and was incubated at 25°C for 30 min. The data are means ± standard deviations from five independent experiments.

FIG 4

Putative −35, −10, and Shine-Dalgarno sequences (A) and amino acid sequence alignment (B) of TCM-HAD and TDK-HAD genes. (A) Putative −35, −10, and Shine-Dalgarno (S.D.) sequences are underlined. The arrow indicates a translation initiation site. (B) Amino acid sequence alignment was performed using ClustalW version 2.1 (http://clustalw.ddbj.nig.ac.jp). The numbers on the right side are the residue numbers for each amino acid sequence. Identical residues and amino acid substitutions with low and high similarities are indicated by asterisks and by dots and double dots, respectively. Amino acid sequences identical to those of internal peptides (see Table S1 in the supplemental material) are underlined.