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Identification and characterization of a novel phosphodiesterase from the metagenome of an Indian coalbed.

Singh DN, Gupta A, Singh VS, Mishra R, Kateriya S, Tripathi AK - PLoS ONE (2015)

Bottom Line: Overexpression of PdeM in E.coli neither affected catabolite respression nor did the recombinant protein hydrolyzed cAMP in vitro, indicating its inability to hydrolyze cAMP.Although Mn2+ was required for the activity of PdeM, but addition of metals (Mn2+ or Fe3+) did not induce oligomerization.PdeM, thus, is a novel representative of new subclass of class III phosphodiesterases.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi-221005, Uttar Pradesh, India.

ABSTRACT
Phosphoesterases are involved in the degradation of organophosphorus compounds. Although phosphomonoesterases and phosphotriesterases have been studied in detail, studies on phosphodiesterases are rather limited. In our search to find novel phosphodiesterases using metagenomic approach, we cloned a gene encoding a putative phosphodiesterase (PdeM) from the metagenome of the formation water collected from an Indian coal bed. Bioinformatic analysis showed that PdeM sequence possessed the characteristic signature motifs of the class III phosphodiesterases and phylogenetic study of PdeM enabled us to identify three distinct subclasses (A, B, and C) within class III phosphodiesterases, PdeM clustering in new subclass IIIB. Bioinformatic, biochemical and biophysical characterization of PdeM further revealed some of the characteristic features of the phosphodiesterases belonging to newly described subclass IIIB. PdeM is a monomer of 29.3 kDa, which exhibits optimum activity at 25°C and pH 8.5, but low affinity for bis(pNPP) as well as pNPPP. The recombinant PdeM possessed phosphodiesterase, phosphonate-ester hydrolase and nuclease activity. It lacked phosphomonoesterase, phosphotriesterase, and RNAse activities. Overexpression of PdeM in E.coli neither affected catabolite respression nor did the recombinant protein hydrolyzed cAMP in vitro, indicating its inability to hydrolyze cAMP. Although Mn2+ was required for the activity of PdeM, but addition of metals (Mn2+ or Fe3+) did not induce oligomerization. Further increase in concentration of Mn2+ upto 3 mM, increased α-helical content as well as the phosphodiesterase activity. Structural comparison of PdeM with its homologs showed that it lacked critical residues required for dimerization, cAMP hydrolysis, and for the high affinity binding of bis(pNPP). PdeM, thus, is a novel representative of new subclass of class III phosphodiesterases.

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Effect of metal ions on the intrinsic fluorescence of PdeM.(A) Effect of Mn2+ and, (B) Effect of Effect of Fe3+ concentrations on the intrinsic tryptophan fluorescence of the purified PdeM protein. Saturation curves were generated from the intrinsic fluorescence data by plotting the change in fluorescence intensity at 340 nm as a function of increasing concentrations of (C) Mn2+ and (D) Fe3+.
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pone.0118075.g005: Effect of metal ions on the intrinsic fluorescence of PdeM.(A) Effect of Mn2+ and, (B) Effect of Effect of Fe3+ concentrations on the intrinsic tryptophan fluorescence of the purified PdeM protein. Saturation curves were generated from the intrinsic fluorescence data by plotting the change in fluorescence intensity at 340 nm as a function of increasing concentrations of (C) Mn2+ and (D) Fe3+.

Mentions: PdeM contains 7 tryptophan residues which are evenly distributed in the protein. Consequently, PdeM shows a fluorescence emission maximum at 340 nm. Increasing the concentration of Mn2+ (0.1 mM to 3.0 mM) led to a gradual decline in the fluorescence upto 53% of the basal level (Fig. 5A). However, a further increase in Mn2+ from 3.0 mM to 4.5 mM led to a drastic reduction in its fluorescence upto 98%. This observation suggested that an increase in Mn2+ concentration upto 3 mM induced a favorable change in the conformation of PdeM protein which increased the enzyme activity whereas further increase in Mn2+ concentration abruptly decreased the fluorescence intensity of the protein (Fig. 5A). In range of 2–3 mM of Mn+2 ion concentrations there is a change in the secondary structure of PdeM, which might result in a change in the tertiary structure as evident by abrupt decrease in fluorescence intensity beyond 3 mM Mn2+. Hence, conformation beyond 3 mM Mn+2 ions may not be favourable for the optimum activity of PdeM. This observation corroborates well with our metal saturation kinetic studies, where maximum phosphodiesterase activity of PdeM was observed at 3 mM Mn2+ and further increase of Mn2+ reduced the PdeM activity (Fig. 2B). In case of Fe3+, the emission declined by 53% even at 0.5 mM concentration while at 1 mM it was reduced by 78% (Fig. 5B). Also, the lack of phosphodiesterase activity in presence of 1 mM Fecl3 (data not shown) is in agreement with our fluorescence data in presence of varying concentrations of Fe3+.


Identification and characterization of a novel phosphodiesterase from the metagenome of an Indian coalbed.

Singh DN, Gupta A, Singh VS, Mishra R, Kateriya S, Tripathi AK - PLoS ONE (2015)

Effect of metal ions on the intrinsic fluorescence of PdeM.(A) Effect of Mn2+ and, (B) Effect of Effect of Fe3+ concentrations on the intrinsic tryptophan fluorescence of the purified PdeM protein. Saturation curves were generated from the intrinsic fluorescence data by plotting the change in fluorescence intensity at 340 nm as a function of increasing concentrations of (C) Mn2+ and (D) Fe3+.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4320098&req=5

pone.0118075.g005: Effect of metal ions on the intrinsic fluorescence of PdeM.(A) Effect of Mn2+ and, (B) Effect of Effect of Fe3+ concentrations on the intrinsic tryptophan fluorescence of the purified PdeM protein. Saturation curves were generated from the intrinsic fluorescence data by plotting the change in fluorescence intensity at 340 nm as a function of increasing concentrations of (C) Mn2+ and (D) Fe3+.
Mentions: PdeM contains 7 tryptophan residues which are evenly distributed in the protein. Consequently, PdeM shows a fluorescence emission maximum at 340 nm. Increasing the concentration of Mn2+ (0.1 mM to 3.0 mM) led to a gradual decline in the fluorescence upto 53% of the basal level (Fig. 5A). However, a further increase in Mn2+ from 3.0 mM to 4.5 mM led to a drastic reduction in its fluorescence upto 98%. This observation suggested that an increase in Mn2+ concentration upto 3 mM induced a favorable change in the conformation of PdeM protein which increased the enzyme activity whereas further increase in Mn2+ concentration abruptly decreased the fluorescence intensity of the protein (Fig. 5A). In range of 2–3 mM of Mn+2 ion concentrations there is a change in the secondary structure of PdeM, which might result in a change in the tertiary structure as evident by abrupt decrease in fluorescence intensity beyond 3 mM Mn2+. Hence, conformation beyond 3 mM Mn+2 ions may not be favourable for the optimum activity of PdeM. This observation corroborates well with our metal saturation kinetic studies, where maximum phosphodiesterase activity of PdeM was observed at 3 mM Mn2+ and further increase of Mn2+ reduced the PdeM activity (Fig. 2B). In case of Fe3+, the emission declined by 53% even at 0.5 mM concentration while at 1 mM it was reduced by 78% (Fig. 5B). Also, the lack of phosphodiesterase activity in presence of 1 mM Fecl3 (data not shown) is in agreement with our fluorescence data in presence of varying concentrations of Fe3+.

Bottom Line: Overexpression of PdeM in E.coli neither affected catabolite respression nor did the recombinant protein hydrolyzed cAMP in vitro, indicating its inability to hydrolyze cAMP.Although Mn2+ was required for the activity of PdeM, but addition of metals (Mn2+ or Fe3+) did not induce oligomerization.PdeM, thus, is a novel representative of new subclass of class III phosphodiesterases.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi-221005, Uttar Pradesh, India.

ABSTRACT
Phosphoesterases are involved in the degradation of organophosphorus compounds. Although phosphomonoesterases and phosphotriesterases have been studied in detail, studies on phosphodiesterases are rather limited. In our search to find novel phosphodiesterases using metagenomic approach, we cloned a gene encoding a putative phosphodiesterase (PdeM) from the metagenome of the formation water collected from an Indian coal bed. Bioinformatic analysis showed that PdeM sequence possessed the characteristic signature motifs of the class III phosphodiesterases and phylogenetic study of PdeM enabled us to identify three distinct subclasses (A, B, and C) within class III phosphodiesterases, PdeM clustering in new subclass IIIB. Bioinformatic, biochemical and biophysical characterization of PdeM further revealed some of the characteristic features of the phosphodiesterases belonging to newly described subclass IIIB. PdeM is a monomer of 29.3 kDa, which exhibits optimum activity at 25°C and pH 8.5, but low affinity for bis(pNPP) as well as pNPPP. The recombinant PdeM possessed phosphodiesterase, phosphonate-ester hydrolase and nuclease activity. It lacked phosphomonoesterase, phosphotriesterase, and RNAse activities. Overexpression of PdeM in E.coli neither affected catabolite respression nor did the recombinant protein hydrolyzed cAMP in vitro, indicating its inability to hydrolyze cAMP. Although Mn2+ was required for the activity of PdeM, but addition of metals (Mn2+ or Fe3+) did not induce oligomerization. Further increase in concentration of Mn2+ upto 3 mM, increased α-helical content as well as the phosphodiesterase activity. Structural comparison of PdeM with its homologs showed that it lacked critical residues required for dimerization, cAMP hydrolysis, and for the high affinity binding of bis(pNPP). PdeM, thus, is a novel representative of new subclass of class III phosphodiesterases.

Show MeSH
Related in: MedlinePlus