A Novel Alkaline Phosphatase/Phosphodiesterase, CamPhoD, from Marine Bacterium Cobetia amphilecti KMM 296.

A novel extracellular alkaline phosphatase/phosphodiesterase from the structural protein family PhoD that encoded by the genome sequence of the marine bacterium Cobetia amphilecti KMM 296 (CamPhoD) has been expressed in Escherichia coli cells. The calculated molecular weight, the number of amino acids, and the isoelectric point (pI) of the mature protein's subunit are equal to 54832.98 Da, 492, and 5.08, respectively. The salt-tolerant, bimetal-dependent enzyme CamPhoD has a molecular weight of approximately 110 kDa in its native state. CamPhoD is activated by Co ²⁺ , Mg ²⁺ , Ca ²⁺ , or Fe ³⁺ at a concentration of 2 mM and exhibits maximum activity in the presence of both Co ²⁺ and Fe ³⁺ ions in the incubation medium at pH 9.2. The exogenous ions, such as Zn ²⁺ , Cu ²⁺ , and Mn ²⁺ , as well as chelating agents EDTA and EGTA, do not have an appreciable effect on the CamPhoD activity. The temperature optimum for the CamPhoD activity is 45 °C. The enzyme catalyzes the cleavage of phosphate mono- and diester bonds in nucleotides, releasing inorganic phosphorus from p-nitrophenyl phosphate (pNPP) and guanosine 5'-triphosphate (GTP), as determined by the Chen method, with rate approximately 150- and 250-fold higher than those of bis-pNPP and 5'-pNP-TMP, respectively. The Michaelis-Menten constant (K _m ), V _max , and efficiency (k _cat /K _m ) of CamPhoD were 4.2 mM, 0.203 mM/min, and 7988.6 S ^-1 /mM; and 6.71 mM, 0.023 mM/min, and 1133.0 S ^-1 /mM for pNPP and bis-pNPP as the chromogenic substrates, respectively. Among the 3D structures currently available, in this study we found only the low identical structure of the Bacillus subtilis enzyme as a homologous template for modeling CamPhoD, with a new architecture of the phosphatase active site containing Fe ³⁺ and two Ca ²⁺ ions. It is evident that the marine bacterial phosphatase/phosphidiesterase CamPhoD is a new structural member of the PhoD family.
Competing Interests: The authors declare no conflict of interest.