**Yulia Noskova 1,\*, Galina Likhatskaya 1, Natalia Terentieva 1, Oksana Son 2, Liudmila Tekutyeva <sup>2</sup> and Larissa Balabanova 1,2,\***


Received: 22 October 2019; Accepted: 19 November 2019; Published: 22 November 2019

**Abstract:** 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 Co2<sup>+</sup>, Mg2<sup>+</sup>, Ca2<sup>+</sup>, or Fe3<sup>+</sup> at a concentration of 2 mM and exhibits maximum activity in the presence of both Co2<sup>+</sup> and Fe3<sup>+</sup> ions in the incubation medium at pH 9.2. The exogenous ions, such as Zn2<sup>+</sup>, Cu2<sup>+</sup>, and Mn2<sup>+</sup>, 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 (Km), Vmax, and efficiency (kcat/Km) 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 Fe3<sup>+</sup> and two Ca2<sup>+</sup> ions. It is evident that the marine bacterial phosphatase/phosphidiesterase CamPhoD is a new structural member of the PhoD family.

**Keywords:** recombinant alkaline phosphatase; bimetal-dependent phosphodiesterase; marine bacterium; *Cobetia amphilecti*; PhoD
