*2.4. Substrate Specificity of CamPhoD*

The study on substrate specificity of CamPhoD has shown that the enzyme catalyzes the cleavage of the phosphate group from deoxy- and ribonucleoside mono-, -di-, and-triphosphates in the following order according to catalytic rate: pNPP ≥ CMP ≥ GTP ≥ UMP ≥ dCMP ≥ AMP ≥ TMP ≥ CTP ≥ GDP ≥ GMP ≥ UDP = CDP ≥ bis-pNPP ≥ 5 -pNP-TMP (Table 2). According to the Chen method [54] of free phosphorus determination, Pi was released from pNPP and GTP under the CamPhoD catalysis approximately 150 and 250 times faster than from bis-pNPP and 5 -pNP-TMP, respectively (Table 2). The CamPhoD activities toward the chromogenic substrates pNPP, bis-pNPP, and 5 -pNP-TMP obtained by the spectrophotometric determination of the p-nitrophenyl concentration were similar. The ability of CamPhoD to cleave the phosphate group of the diesters bis-pNPP and 5 -pNP-TMP, as well as different phosphate monoesters, allowed us to assign this enzyme to the bifunctional phosphatase/phosphodiesterase [9,10,16–20,55]. For comparison, the binuclear zinc phosphodiesterase ZiPD from *E. coli* possessed activity towards phosphodiester bonds of bis-pNPP and 5 -pNP-TMP only, and did not catalyze the cleavage of other phosphates, such as AMP, ADP, ATP, cyclic phosphates, or nucleic acids [45]. Despite a wide variety of nucleoside mono-, di-, and triphosphates used as substrates, CamPhoD did not catalyze the cleavage of the λ DNA, plasmid pUC19 DNA (Thermo Scientific, Vilnus, Lithuania), oligonucleotides (Evrogen, Moscow), or c-di-GMP (Sigma) up to inorganic phosphorus Pi (Table 2), similar to the phosphodiesterase PhoD from *B. subtilis* [15].


**Table 2.** Substrate specificity of the alkaline phosphatase/phosphodiesterase CamPhoD from *Cobetia amphilecti* KMM 296 \*.

\* These results were obtained using a molybdate reagent with ascorbic acid [54]. Various substrates were added to the reaction mixture at 2–15 mM concentrations. For each substrate, a control consisting of a specific substrate and a buffer containing 25 mM Tris-HCl (pH 9.0), 2 mM CoCl2, and 2 mM FeCl3 was used.
