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

Abstract

A novel extracellular alkaline phosphatase/phosphodiesterase from the structural protein family PhoD that encoded by the genome sequence of the marine bacterium <i>Cobetia amphilecti</i> KMM 296 (CamPhoD) has been expressed in <i>Escherichia coli</i> 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 Co²⁺, Mg²⁺, Ca²⁺, or Fe³⁺ at a concentration of 2 mM and exhibits maximum activity in the presence of both Co²⁺ and Fe³⁺ ions in the incubation medium at pH 9.2. The exogenous ions, such as Zn²⁺, Cu²⁺, and Mn²⁺, 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 <i>Bacillus subtilis</i> enzyme as a homologous template for modeling CamPhoD, with a new architecture of the phosphatase active site containing Fe³⁺ and two Ca²⁺ ions. It is evident that the marine bacterial phosphatase/phosphidiesterase CamPhoD is a new structural member of the PhoD family.