A Novel Alkaline Phosphatase/Phosphodiesterase, CamPhoD, from Marine Bacterium <i>Cobetia amphilecti</i> KMM 296
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 mol...
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MDPI AG
2019-11-01
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| Series: | Marine Drugs |
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| author | Yulia Noskova Galina Likhatskaya Natalia Terentieva Oksana Son Liudmila Tekutyeva Larissa Balabanova |
| author_facet | Yulia Noskova Galina Likhatskaya Natalia Terentieva Oksana Son Liudmila Tekutyeva Larissa Balabanova |
| author_sort | Yulia Noskova |
| collection | DOAJ |
| description | 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 Co<sup>2+</sup>, Mg<sup>2+</sup>, Ca<sup>2+</sup>, or Fe<sup>3+</sup> at a concentration of 2 mM and exhibits maximum activity in the presence of both Co<sup>2+</sup> and Fe<sup>3+ </sup>ions in the incubation medium at pH 9.2. The exogenous ions, such as Zn<sup>2+</sup>, Cu<sup>2+</sup>, and Mn<sup>2+</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 (K<sub>m</sub>), V<sub>max</sub>, and efficiency (k<sub>cat</sub>/K<sub>m</sub>) of CamPhoD were 4.2 mM, 0.203 mM/min, and 7988.6 S<sup>-1</sup>/mM; and 6.71 mM, 0.023 mM/min, and 1133.0 S<sup>-1</sup>/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 Fe<sup>3+</sup> and two Ca<sup>2+</sup> ions. It is evident that the marine bacterial phosphatase/phosphidiesterase CamPhoD is a new structural member of the PhoD family. |
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| issn | 1660-3397 |
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| publishDate | 2019-11-01 |
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| spelling | doaj.art-48bf917c8d2044909b943b340878218c2022-12-22T04:01:16ZengMDPI AGMarine Drugs1660-33972019-11-01171265710.3390/md17120657md17120657A Novel Alkaline Phosphatase/Phosphodiesterase, CamPhoD, from Marine Bacterium <i>Cobetia amphilecti</i> KMM 296Yulia Noskova0Galina Likhatskaya1Natalia Terentieva2Oksana Son3Liudmila Tekutyeva4Larissa Balabanova5Laboratories of Marine Biochemistry and Bioassays and Mechanisms of Action of Biologically Active Substances, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, the Russian Academy of Sciences, 690022 Vladivostok, RussiaLaboratories of Marine Biochemistry and Bioassays and Mechanisms of Action of Biologically Active Substances, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, the Russian Academy of Sciences, 690022 Vladivostok, RussiaLaboratories of Marine Biochemistry and Bioassays and Mechanisms of Action of Biologically Active Substances, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, the Russian Academy of Sciences, 690022 Vladivostok, RussiaSchool of Economics and Management of Far East Federal University, 690950 Vladivostok, RussiaSchool of Economics and Management of Far East Federal University, 690950 Vladivostok, RussiaLaboratories of Marine Biochemistry and Bioassays and Mechanisms of Action of Biologically Active Substances, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, the Russian Academy of Sciences, 690022 Vladivostok, RussiaA 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 Co<sup>2+</sup>, Mg<sup>2+</sup>, Ca<sup>2+</sup>, or Fe<sup>3+</sup> at a concentration of 2 mM and exhibits maximum activity in the presence of both Co<sup>2+</sup> and Fe<sup>3+ </sup>ions in the incubation medium at pH 9.2. The exogenous ions, such as Zn<sup>2+</sup>, Cu<sup>2+</sup>, and Mn<sup>2+</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 (K<sub>m</sub>), V<sub>max</sub>, and efficiency (k<sub>cat</sub>/K<sub>m</sub>) of CamPhoD were 4.2 mM, 0.203 mM/min, and 7988.6 S<sup>-1</sup>/mM; and 6.71 mM, 0.023 mM/min, and 1133.0 S<sup>-1</sup>/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 Fe<sup>3+</sup> and two Ca<sup>2+</sup> ions. It is evident that the marine bacterial phosphatase/phosphidiesterase CamPhoD is a new structural member of the PhoD family.https://www.mdpi.com/1660-3397/17/12/657recombinant alkaline phosphatasebimetal-dependent phosphodiesterasemarine bacterium<i>cobetia amphilecti</i>phod |
| spellingShingle | Yulia Noskova Galina Likhatskaya Natalia Terentieva Oksana Son Liudmila Tekutyeva Larissa Balabanova A Novel Alkaline Phosphatase/Phosphodiesterase, CamPhoD, from Marine Bacterium <i>Cobetia amphilecti</i> KMM 296 Marine Drugs recombinant alkaline phosphatase bimetal-dependent phosphodiesterase marine bacterium <i>cobetia amphilecti</i> phod |
| title | A Novel Alkaline Phosphatase/Phosphodiesterase, CamPhoD, from Marine Bacterium <i>Cobetia amphilecti</i> KMM 296 |
| title_full | A Novel Alkaline Phosphatase/Phosphodiesterase, CamPhoD, from Marine Bacterium <i>Cobetia amphilecti</i> KMM 296 |
| title_fullStr | A Novel Alkaline Phosphatase/Phosphodiesterase, CamPhoD, from Marine Bacterium <i>Cobetia amphilecti</i> KMM 296 |
| title_full_unstemmed | A Novel Alkaline Phosphatase/Phosphodiesterase, CamPhoD, from Marine Bacterium <i>Cobetia amphilecti</i> KMM 296 |
| title_short | A Novel Alkaline Phosphatase/Phosphodiesterase, CamPhoD, from Marine Bacterium <i>Cobetia amphilecti</i> KMM 296 |
| title_sort | novel alkaline phosphatase phosphodiesterase camphod from marine bacterium i cobetia amphilecti i kmm 296 |
| topic | recombinant alkaline phosphatase bimetal-dependent phosphodiesterase marine bacterium <i>cobetia amphilecti</i> phod |
| url | https://www.mdpi.com/1660-3397/17/12/657 |
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