The mechanism of action of the manganese -dependent phosphotriesterase from Sphingobium sp. strain TCM1 that is capable of hydrolyzing organophosphate flame retardants was determined. The enzyme was shown to hydrolyze the R-P-enantiorner of O-methyl O-cydohexyl p-nitrophenyl thiophosphate with net inversion of configuration and without the formation of a covalent reaction intermediate. These results demonstrate that the enzyme catalyzes the hydrolysis of substrates by activation of a nucleophilic water molecule for direct attack at the phosphorus center.