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

• Published in: Marine drugs Vol. 17; no. 12; p. 657
• Main Authors: Noskova, Yulia, Likhatskaya, Galina, Terentieva, Natalia, Son, Oksana, Tekutyeva, Liudmila, Balabanova, Larissa
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 22-11-2019; MDPI
• Subjects: Alkaline phosphatase; Alkaline Phosphatase - chemistry; Alkaline Phosphatase - genetics; Alkaline Phosphatase - isolation & purification; Alkaline Phosphatase - metabolism; Alzheimers disease; Amino acids; Approximation; Aquatic Organisms - enzymology; Aquatic Organisms - genetics; Bacteria; bimetal-dependent phosphodiesterase; Bimetals; Binding sites; Calcium; Calcium ions; Carbon dioxide; Chelates; Chelating agents; Chelation; Cobalt; Cobetia; cobetia amphilecti; Copper; E coli; Enzyme Assays; Enzymes; Ethylenediaminetetraacetic acids; Extracellular; Ferric ions; Genomes; Halomonadaceae - enzymology; Halomonadaceae - genetics; Homology; Incubation period; Ions; Iron; Kinases; Magnesium; marine bacterium; Metabolism; Metabolites; Mineralization; Molecular weight; Nucleotide sequence; Nucleotides; p-Nitrophenylphosphate; Peptides; phod; Phosphatase; Phosphates; Phosphodiesterase; Phosphodiesterase I - chemistry; Phosphodiesterase I - genetics; Phosphodiesterase I - isolation & purification; Phosphodiesterase I - metabolism; Phosphorus; Protein Structure, Tertiary; Proteins; recombinant alkaline phosphatase; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Salinity tolerance; Signal transduction; Structural members; Substrates; Zinc; bimetal-dependent phosphodiesterase; PhoD; marine bacterium; Cobetia amphilecti; recombinant alkaline phosphatase
• ISSN: 1660-3397; 1660-3397
• DOI: 10.3390/md17120657

A novel extracellular alkaline phosphatase/phosphodiesterase from the structural protein family PhoD that encoded by the genome sequence of the marine bacterium
KMM 296 (CamPhoD) has been expressed in
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
, Mg
, Ca
, or Fe
at a concentration of 2 mM and exhibits maximum activity in the presence of both Co
and Fe
ions in the incubation medium at pH 9.2. The exogenous ions, such as Zn
, Cu
, and Mn
, 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
), V
, and efficiency (k
/K
) of CamPhoD were 4.2 mM, 0.203 mM/min, and 7988.6 S
/mM; and 6.71 mM, 0.023 mM/min, and 1133.0 S
/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
enzyme as a homologous template for modeling CamPhoD, with a new architecture of the phosphatase active site containing Fe
and two Ca
ions. It is evident that the marine bacterial phosphatase/phosphidiesterase CamPhoD is a new structural member of the PhoD family.