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

Liudmila Tekutyeva,Yulia Noskova,Larissa Balabanova,Oksana Son,Natalia Terentieva,Galina Likhatskaya

doi:10.3390/md17120657

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

Highlights

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 [1,2,3]
Natural and recombinant alkaline phosphatases isolated from marine bacteria have been described, but information about their alkaline phosphodiesterases is still lacking [6,12,13,14,15]
The CamPhoD phosphodiesterase activity at the cleavage of bis-p-nitrophenyl phosphate (pNPP) was 0.3 U/mg. The isolation of this enzyme confirmed the ability of C. amphilecti to produce the functionally active alkaline bifunctional phosphatase/phosphodiesterase CamPhoD with a calculated molecular weight of 54839.8 kDa for the mature protein, without the 33-letter leader peptide, according to the Simple Modular Architecture

Summary

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 [1,2,3]. 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. Natural and recombinant alkaline phosphatases isolated from marine bacteria have been described, but information about their alkaline phosphodiesterases is still lacking [6,12,13,14,15]

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Results

Conclusion