Abstract
Using a screening procedure developed for detection of phytate hydrolysing enzymes, the gene agpE encoding glucose-1-phosphatase was cloned from an Enterobacter cloacae VKPM B2254 plasmid library. Sequence analysis revealed 78% identity on nucleotide and 79% identity on peptide level to Escherichia coli glucose-1-phosphatase characterising the respective gene product as a representative of acid histidine phosphatases harbouring the RH(G/N)RXRP motif. The purified recombinant protein displayed maximum specific activity of 196 U mg−1 protein against glucose-1-phosphate but was also active against other sugar phosphates and p-nitrophenyl phosphate. High-performance ion chromatography of hydrolysis products revealed that AgpE can act as a 3-phytase but is only able to cleave off the third phosphate group from the myo-inositol sugar ring. Based on sequence comparison and catalytic behaviour against phytate, we propose to classify bacterial acid histidine phosphatases/phytases in the three following subclasses: (1) AppA-related phytases, (2) PhyK-related phytases and (3) Agp-related phytases. A distinguished activity of 32 U mg−1 of protein towards myo-inositol-hexa-phosphate, which is two times higher than that of E. coli Agp, suggests that possibly functional differences in terms of phytase activity between Agp- and AppA-like acid histidine phosphatases are fluent.
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Acknowledgements
We thank Dr. Sineoky, Moscow, for providing the Enterobacter cloacae strain used in this study and Mrs. Christiane Müller, Berlin, for excellent technical assistance. We are much obliged to Kathleen Smith, Paris and the unknown reviewers for many valuable suggestions improving the manuscript. Dr. Andreas Weihe, Berlin, is especially thanked for his support in using the PAUP package for construction of the evolutionary tree. We are very grateful to BMBF for the continuous financial support given in the framework of the exchange project RUS 01/235.
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253_2005_24_MOESM1_ESM.pdf
Gene organization in recombinant plasmids phy 5 and phy 6. Size and structure of subclones phy 4, phy 11 and phy 12 are also shown. Bold arrows indicate the direction of open reading frames (PDF 15kb)
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Multiple alignment of homologs of the E. cloacae Agp. Conserved histidine and phosphatase family motifs, residues with functional importance for Agp and specific deletions, are indicated (PDF 12kb)
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SDS-PAGE of AgpE fractions after His-Tag purification. From left to right, a std, molecular mass (Fermentas); L, cell lysate; D, run through; W, washing buffer; W25, column eluant with 25 mM imidazole; 1–5, eluants with increasing imidazole concentration (50–100 mM); b 6, 100 mM imidazole; std, molecular mass (Fermentas); 7–10, eluants with 100 mM imidazole; 11–13, eluants with 150 mM imidazole. The arrows indicate glucose-1-phosphatase (PDF 21kb)
253_2005_24_MOESM4_ESM.pdf
Molecular weight chromatography of native AgpE at different pH. Column calibration was performed with gel filtration chromatography standard 6 vials (BioRad) containing one thyroglobin 670 kDa, two bovine γ-globulin 158 kDa, three chicken ovalbumin 44 kDa, four equine myoglobin 17 kDa, five Vitamin B12 1.35 kDa in Tris–HCl pH 7.5 and 150 mM NaCl (a) and 100 mM acetate buffer with 150 mM NaCl pH 6.0 (c). Twenty-microliter purified AgpE were loaded under same conditions at pH 6.0 (b) and at pH 7.5 (d). The absorptions were measured in microabsorption units. The apparent molecular masses for fraction 8 (pH 6.0) and fraction 10 (pH 7.5) were calculated according to their R f values as to be 125.9 kDa at pH 6.0 and 82.5 kDa at pH 7.5, respectively (PDF 255kb)
253_2005_24_MOESM5_ESM.pdf
Characterization of the fractions obtained after native Gel chromatography at pH 7.5 (see Electronic Supplementary Material, Fig. S4). a Western blot after SDS-PAGE with His-tag specific antibodies; Std, molecular mass (Fermentas). b Glucose-1-phosphatase activities of fractions after column chromatography (PDF 51kb)
253_2005_24_MOESM6_ESM.pdf
Effect of pH on enzyme activity of AgpE using substrates glucose-1-phosphate (■), InsP6 (▲) and pNPP (♦). The bars represent the standard deviation (n=3) (PDF 44kb)
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Herter, T., Berezina, O.V., Zinin, N.V. et al. Glucose-1-phosphatase (AgpE) from Enterobacter cloacae displays enhanced phytase activity. Appl Microbiol Biotechnol 70, 60–64 (2006). https://doi.org/10.1007/s00253-005-0024-8
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DOI: https://doi.org/10.1007/s00253-005-0024-8