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

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.

Enzyme promiscuity in natural environments: alkaline phosphatase in the ocean

“…It is important to note that three APase gene families are categorically known in prokaryotes i.e., PhoA, PhoD, and PhoX, where latter two are predicted to be more abundant in ocean than PhoA [39]. PhoD and PhoX have specificity toward diester substrate in addition to monoester substrate, whereas PhoA has specificity toward monoester substrate only [39][40][41][42][43]. In addition, there is no reported evidence of PhoD and PhoX phosphatases exhibiting triesterase and sulfatase activity.…”

Are the Closely Related Cobetia Strains of Different Species?

“…The DNA/RNA endonuclease belonging to the structural cl00089: NUC superfamily can non-specifically cleave both double- and single-stranded DNA and RNA, whose domain may be present in phosphodiesterases [ 23 ]. Thus, all these enzymes are fundamental for bacteria survival [ 24 , 25 , 26 , 27 , 28 , 29 ]. If the strains Cobetia sp.…”

The Response of N2-Fixing Bacteria To PH Changes, Temperature, And Elevated Levels of CO2 Can Depend On Their Nutrient Status

“…amphilecti KMM 296) the optimal APA is within a range of 40 to 45 ºC [76,78,81]. To date, this is the first study reporting the in vivo temperature effect of the APA in marine N2-fixing bacteria (Figure 4B), considering that the stability of the enzyme, the velocity of organic phosphate breakdown, and enzyme-substrate affinity are controlled by temperature [82].…”

“…Cobetia amphilecti KMM 296) show the dramatical inhibition of APA with acidification, possibly due to loss of the stability of the enzyme [76][77][78]. Interestingly, APA was more sensitive for cyanobacterial strains, being highly inhibited below pH 7, while heterotrophic bacteria were able to maintain higher rates of APA in a range from 6.5-8…”