**1. Introduction**

Alkaline phosphatases are widely distributed in marine bacteria, which release inorganic phosphate (Pi) from phosphorus-containing compounds dissolved in the ocean and utilize them for their own growth and reproduction [1–3]. Globally, marine bacteria and diatoms have been shown to store and concentrate Pi and then release it into the local marine environment using phosphatase (Pho) activity, thus biologically inducing and controlling phosphorite and apatite nucleation [4,5]. Moreover, the enzymes from marine sources have flexible molecular structures, and therefore work better at ambient or lower temperatures, which opens the possibility to decrease temperatures of production processes for their application in biotechnology [6–9].

Currently, three large families of prokaryotic alkaline phosphatases are known, namely PhoA, PhoD, and PhoX. They differ from each other in structure, substrate specificity, and the dependence on different metal ions for the manifestation of their activities [10]. It has been shown that the PhoD-like phosphatases belonging to the phosphatase/phosphodiesterase family more commonly occur in marine and soil bacteria than PhoA and PhoX [11]. This is due to the presence in the environment of available sources of phosphorus and cofactors in their habitat. It has been proven that the PhoD family is common in the bacteria living in phosphorus- and metal-depleted conditions [3,5,11]. Previously, natural and recombinant alkaline phosphatases isolated from marine bacteria have been described, but information about their alkaline phosphodiesterases is still lacking [6,12–15].

There are many families of phosphodiesterases, which include phospholipases C and D, autotaxin, sphingomyelin phosphodiesterase, DNases, RNases, and restriction endonucleases. However, phosphodiesterases usually refer to the cyclic nucleotide phosphodiesterases degrading cyclic adenosine and guanosine monophosphates (cAMP and cGMP). According to the primary structure and differences in the catalytic domains, they are divided into three known classes: (1) the eukaryotic enzymes; (2) enzymes such as phosphodiesterases from the yeast *Dictyostelium* and the bacteria *Vibrio*; and (3) the bacterial enzymes homologous to purple acid phosphatases and dimetallophosphoesterases, including the three subclasses A, B, and C [16]. The bacterial phosphodiesterases were isolated and characterized from *Aphanothece halophytica* [9], *Delftia acidovorans* [17], *Sphingobium* sp. TCM1 [18], *E. coli* [19], *B. subtilis* [20], as well as a novel unclassified enzyme from the metagenome of an Indian coalbed [16]. All these metal-dependent phosphodiesterases showed maximal activities in the alkaline pH range, and needed different metal ions, such as Ca2<sup>+</sup>, Zn2<sup>+</sup>, Mg2<sup>+</sup>, or Mn2+, for their catalytic activity. The isolated phosphodiesterases were capable of cleaving phosphoric acid residues from specific substrates, such as Bis-*p*-nitrophenyl phosphate (Bis-pNPP) and thymidine-5 -monophosphate-*p*-nitrophenylester (5 -pNP-TMP), which are mostly used as DNA models for studies of phosphodiester hydrolysis [16,21]. It has been previously assumed that the role of PhoD-like enzymes is to participate in the nucleic acid exchange in cells during the main metabolism, taking into account their ability to hydrolyze the phosphodiester bonds [22].

Among three types of phosphoester bonds existing in nature (mono-, di-, and triester), the phosphodiester bond is exceptionally stable, with a half-life of approximately 3 <sup>×</sup> 10<sup>7</sup> years at a moderate temperature and a neutral pH, while an acceleration of its cleavage up to 1016-fold in biological processes can be achieved through enzymatic hydrolysis of this bond by the highly specialized metalloenzymes, such as nucleases and phosphoesterases [23,24]. However, finding and exploring novel enzymes with phosphoesterase activity is still a challenge in biotechnology because of some their inherent limitations, such as undesirable selectivity, difficulties in extraction or synthesis, high cost, narrow functional temperature, and pH range [25]. The phosphoesterase function in nature may be related to hydrolyzing a wide range of biomolecules (proteins, nucleic acids, and lipids) implicated in DNA repair, post-translational modification, biomineralization, and energy metabolism, as well as in signal transduction through regulation of the circulation of secondary metabolites, particularly free nucleotides and their analogues [26,27]. The phosphodiesterase families are mostly considered to have a common catalytic domain pocket, with the universal mechanism of nucleophilic attack to control the intracellular levels of cyclic nucleotides, and to be regulators of many physiological and pathophysiological processes [28]. Due to their important role in intracellular signal transduction and the possibility of finding their exact subcellular localization, phosphodiesterases are considered very attractive pharmacological targets [29,30]. Therefore, there is a growing interest in finding ways to disrupt, block, or manipulate quorum sensing (QS) signaling in bacteria [29]. The producers of QS signals have been found among both the free-living and associated marine bacteria inhabiting

invertebrates and algae [31]. Consequently, they are promising sources for new bioactive compounds, such as the QS modulators or inhibitors [29].

The QS-related phosphodiesterases of marine origin have yet to be investigated. However, two alkaline phosphatases of the juvenile *Euprymna scolopes* light organ were found to play an active role in dephosphorylating lipid A of the luminous marine bacterium *Vibrio fischeri,* which changes its signaling properties in relation to the host tissues during their symbiotic colonization [32]. The PhoA alkaline phosphatase (CmAP) of the marine bacterium *C. amphilecti* KMM 296 (Collection of Marine Microorganisms, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, the Russian Academy of Sciences (PIBOC FEB RAS)) isolated from the coelomic fluid of the mussel, *Crenomytilus grayanus,* was suggested to promote the host mollusk shell's mineralization and biofilm regulation of many species of food-derived pathogens [6,7,12]. The mechanism of the CmAP biological action is still unclear and remains under investigation.

Thus, it has been recently shown that the biological role of alkaline phosphatases is more complex and broader than previously assumed. Alkaline phosphatases appear to be involved in major cellular events, such as the regulation of protein phosphorylation, cell growth, apoptosis, and cellular migration [32–34]. Therefore, most human conditions or diseases are accompanied by a change in the level of alkaline phosphatase expression, which is the basis of diagnostics. A newly discovered function of alkaline phosphatases is in maintaining tissue and organ homeostasis by inactivation of bacterial lipopolysaccharides (LPS), and by regulation of cell secretion, microbiome and tumor behavior, and possibly detoxication of hyperphosphorylated extracellular tau proteins, which play a key role in progression of Alzheimer's disease [32–34]. Recently, bovine and human intestinal recombinant alkaline phosphatases underwent clinical trials related to inactivating LPS and preventing inflammation for the treatment of surgical diseases and metabolic disorders [33–35]. It is possible that the search for marine enzymes with dephosphorylating activity and the study of their mechanism of action will also present an application for the new treatment.

The genome sequence analysis of *C. amphilecti* KMM 296 has shown that the bacterium produces not only the highly active alkaline phosphatase CmAP, belonging to the PhoA family (GenBank ID: WP\_084589490.1), but also the functionally active PhoD-like phosphatase/phosphodiesterase (GenBank ID: WP\_043333989.1), with a novel structure and properties [6,12,36]. This article presents the results of isolation of the gene encoding for the PhoD-like protein from *C. amphilecti* KMM 296, and in producing the recombinant enzyme CamPhoD with the alkaline phosphatase and phosphodiesterase activities and properties.
