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.

Tris(2-chloroethyl) and tris(1,3-dichloro-2-propyl) phosphates are chlorinated persistent flame retardants that have recently emerged as environmental pollutants. Two bacterial strains that can degrade the compounds when they are the sole phosphorus sources have been isolated and identified as members of the sphingomonads. The strains can be useful for the bioremediation of environments contaminated with these compounds.The chlorinated organophosphorus (OP) compounds tris(2-chloroethyl) phosphate (TCEP) and tris(1,3-dichloro-2-propyl) phosphate (TDCPP) are widely used as flame retardants mainly in a range of plastic foams, resins, and latexes and in the production of liquid unsaturated polyester resins, respectively (21). Their widespread use has led to their contamination of various environments, including indoor air and house dust (9, 15), surface water (2), ground water (4), rain and snow (11), and sediment (6), in concentrations ranging from ng/liter up to several g/liter. Particularly, a higher contamination level was observed in leachates and raw water of waste disposal sites (7). Furthermore, they were also detected in drinking water (21) because they are hard to eliminate not only in the environment but also in sewage treatment plants (1). Many studies have shown several toxic effects of the compounds. TCEP has been shown to cause adverse effects on brain, liver, and kidney and on the fertility of male rats and mice (21). In addition, TCEP has produced tumors at various organ sites (19) and has also been suspected to possess carcinogenicity (10); although there are contradictory results on its carcinogenicity (3), TCEP has been shown to inhibit the expression of cell cycle regulatory proteins, DNA synthesis, and cell numbers (12). TDCPP has exhibited genotoxicity in several in vitro assays conducted in prokaryotic and eukaryotic cells (14) and produced some indications of carcinogenicity (21). These observations have prompted the recognition of potential ecological and human health concerns.Many bacteria and fungi capable of degrading OP pesticides and insecticides, such as parathion and chlorpyrifos, have been discovered, isolated, and characterized (13). In contrast, few studies have been conducted on microbial degradation of chlorinated OP flame retardants, despite their persistence and potential, nonnegligible toxicity. Thus far, there has been no report of the isolation of bacteria capable of degrading TCEP and TDCPP.We recently demonstrated that these compounds were rapidly degraded in two enrichment bacterial cultures, named 45D and 67E, obtained by using TCEP and TDCPP as sole phosphorus sources (16). We therefore attempted to isolate bacteria capable of degrading the compounds from each enrichment culture. Five hundred microliters of 45D stored at 4°C was transferred to 100 ml of A-Cl medium containing 20 M TDCPP (Wako Pure Chemical, Japan) as the sole phosphorus source (16). Medium A-Cl is a minimal medium composed of 10 g/liter glucose, 5.22 g/liter 3-morpholinepropanesulfonic acid, 1 g of (NH...

D-Amino acid oxidase (DAO) is a flavin enzyme that catalyzes the oxidative deamination of d-amino acids. This enzyme has been studied extensively both biochemically and structurally as a model for the oxidase-dehydrogenase class of flavoproteins. This enzyme also has various applications, such as the determination of d-amino acids and production of building blocks for a number of pharmaceuticals. DAO has been found mainly in eukaryotic organisms and has been suggested to play a significant role in various cellular processes, one of which includes neurotransmission in the human brain. In contrast, this enzyme has not been identified in prokaryotic organisms. Some studies have recently identified and characterized DAO enzyme in some actinobacteria. In addition, a genome database search reveals a wide distribution of DAO homologous genes in this bacterial group. The bacterial DAOs characterized so far have certain distinct properties in comparison to eukaryotic DAOs. These enzymes also exhibit some important applicable properties, suggesting that bacteria could be used as a source for obtaining novel and useful DAOs. The physiological function of bacterial DAO have been proposed to include the degradation of non-canonical d-amino acids released from cell wall, but is still largely unknown and need to be studied in depth.