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bioRxiv · 10.1101/2025.03.21.644484

A universal polyphosphate kinase powers in vitro transcription

Matsumoto, R.·Watanabe, T.·Yamazaki, E.·Kagawa, A.·Longo, L. M.·Matsuura, T.
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Abstract

Polyphosphate kinases (PPKs) catalyze phosphoryl transfer between polyphosphates and nucleotides. Polyphosphates are a cost-effective source of phosphorylating power, making polyphosphate kinases an attractive enzyme for nucleotide production and regeneration. However, at present, applications that require the simultaneous utilization of diverse nucleotides, such as RNA transcription, remain out of reach due to the restricted substrate profiles of known PPK enzymes. Here, we present the discovery and characterization of a universal PPK capable of efficiently phosphorylating all eight common ribonucleotides: purines and pyrimidines, monophosphates and diphosphates to triphosphates. Under optimal reaction conditions, approximately 70% triphosphate conversion was observed for all common ribonucleotides, with only limited over-phosphorylation. At elevated temperatures, however, production of adenine-capped polyphosphates up to a 30-mer was achieved. An analysis of mutant and chimeric enzymes revealed a rugged functional landscape, particularly for non-adenine nucleotides. Finally, we demonstrated the biotechnological potential of a universal PPK enzyme with a one-pot assay for PPK-powered in vitro transcription.

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Matsumoto, R., Watanabe, T., Yamazaki, E., Kagawa, A., Longo, L. M., Matsuura, T.. 2025-03-21. A universal polyphosphate kinase powers in vitro transcription. https://doi.org/10.1101/2025.03.21.644484

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