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Identification, expression and biochemical characterization of AMP phosphorylases from extreme environments
Nucleotide analogs are interesting pharmaceutical intermediates as they represent the active form of nucleoside analog drugs, that are used in the treatment of cancer or viral infections. They are used as precursors in the preparation of artificial oligonucleotides for therapeutic or diagnostic use....
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AUC Knowledge Fountain
2019
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| Summary: | Nucleotide analogs are interesting pharmaceutical intermediates as they represent the active form of nucleoside analog drugs, that are used in the treatment of cancer or viral infections. They are used as precursors in the preparation of artificial oligonucleotides for therapeutic or diagnostic use. Enzymes as active biocatalysts offer numerous advantages over traditional chemical processes with respect to high process selectivity and efficiency. Recently, adenosine-5’-monophosphate phosphorylase (AMP-P) was identified to catalyze the reversible phosphorolysis of nucleotides into nucleobase and ribose-1,5-bisphosphate. Therefore, it is an attractive and promising biocatalyst in the synthesis of nucleotides and their analogs. The availability of enzymes with wide substrate spectra is an important prerequisite to produce a variety of modified nucleotides in enzymatic processes. Consequently, interesting AMP-Ps were produced and characterized concerning their substrate spectra. Red sea metagenomic data and sequences of the National Center for Biotechnology Information database were screened for putative AMP-Ps. Phylogenetic analysis was performed to identify interesting candidates. Sixteen AMP-Ps were chosen from different phylogenetic clusters for gene synthesis based on the differences in the active residues, phylogenetic distance, and variability of their isolation extreme environment. AMP-Ps was successfully expressed in E. coli and purified. Expression conditions were optimized to reach higher amounts of soluble protein. Activity assays were performed with six substrates; AMP, CMP, GMP, UMP, adenosine, and uridine. For AMP-P of Thermococcus khodakarensis (Am01) published data were confirmed. Additionally, Am03 and Am15 showed similar substrate spectra (an activity with AMP, GMP, and UMP) and are putative AMP-P. Their activity increased with increasing temperature which is in good accordance with the temperature optimum of the donor organisms (Thermosphaera aggregans, Thermofilum pendens respectively). The other identified proteins could be putative pyrimidine/purine nucleoside phosphorylase as they show a phosphorylase activity against adenosine and uridine. Am12 from ATII-LCL is an interesting candidate to be further analyzed as it is derived from an extreme environment, has activity towards adenosine and uridine nucleosides and low activity against AMP. In this study, we confirmed the hypothesis that extreme environments can also provide cytoplasmic enzymes with novel characteristics. |
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