Enzymatic characterization and regulation of gene expression of PhoK alkaline phosphatase in Sphingobium sp. strain TCM1.

Sphingobium sp. strain TCM1 can significantly degrade chlorinated organophosphorus flame retardants, such as tris(2-chloroethyl) phosphate. The PhoK of strain TCM1 (Sb-PhoK) is the main alkaline phosphatase (APase) that catalyzes the last step in the degradation pathway. Here, we purified and characterized Sb-PhoK produced in E. coli, and analyzed the regulation of Sb-phoK gene expression in strain TCM1. The recombinant Sb-PhoK was produced in the mature form, lacking a putative signal peptide, and formed a homodimer. Purified Sb-PhoK exhibited 384 U/mg of specific activity at 37 °C. The optimum temperature was 50 °C, and Sb-PhoK was completely inactivated when incubated at 60 °C for 10 min. The optimum pH was 10, with stability observed at pH 6.0-10.5. Sb-PhoK was suggested to contain two Ca2+ and one Zn2+ per subunit, but excess addition of Zn2+ into the reaction mixture markedly inhibited the enzyme activity. Sb-PhoK showed phosphatase activity against various phosphorylated compounds, except for bis(p-nitrophenyl) phosphate, indicating that it is a phosphomonoesterase with broad substrate specificity. The Km and kcat for p-nitrophenyl phosphate were 2.31 mM and 1270 s-1, respectively, under optimal conditions. The enzyme was strongly inhibited by vanadate, dithiothreitol, and SDS, but was highly resistant to urea and Triton X-100. Sb-phoK gene expression was regulated by the inorganic phosphate concentration in culture medium, and was induced at a low inorganic phosphate concentration. The deletion of Sb-phoB gene resulted in no induction of Sb-phoK gene even at a low inorganic phosphate concentration, confirming that Sb-PhoK is a member of Pho regulon.

An atypical phosphodiesterase capable of degrading haloalkyl phosphate diesters from Sphingobium sp. strain TCM1.

Sphingobium sp. strain TCM1 can degrade tris(2-chloroethyl) phosphate (TCEP) to inorganic phosphate and 2-chloroethanol. A phosphotriesterase (PTE), phosphodiesterase (PDE) and phosphomonoesterase (PME) are believed to be involved in the degradation of TCEP. The PTE and PME that respectively catalyze the first and third steps of TCEP degradation in TCM1 have been identified. However, no information has been reported on a PDE catalyzing the second step. In this study, we identified, purified, and characterized a PDE capable of hydrolyzing haloalkyl phosphate diesters. The final preparation of the enzyme had a specific activity of 29 µmol min-1 mg-1 with bis(p-nitrophenyl) phosphate (BpNPP) as the substrate. It also possessed low PME activity with p-nitrophenyl phosphate (pNPP) as substrate. The catalytic efficiency (k cat/K m) with BpNPP was significantly higher than that with pNPP, indicating that the enzyme prefers the organophosphorus diester to the monoester. The enzyme degraded bis(2,3-dibromopropyl) phosphate, bis(1,3-dichloro-2-propyl) phosphate and bis(2-chloroethyl) phosphate, suggesting that it is involved in the metabolism of haloalkyl organophosphorus triesters. The primary structure of the PDE from TCM1 is distinct from those of typical PDE family members and the enzyme belongs to the polymerase and histidinol phosphatase superfamily.