Journal Article

PUBDB-2025-04021

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Structure-guided engineering of a polyphosphate kinase 2 class III from an Erysipelotrichaceae bacterium to produce base-modified purine nucleotides

Mitton-Fry, R. M. ; Rasche, R. ; Lawrence-Dörner, A.-M. ; Eschenbach, J. ; Tekath, A. ; Rentmeister, A. ; Kümmel, D. ; Cornelissen, N. V. (Corresponding author)

2025
The Royal Society of Chemistry Cambridge

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Please use a persistent id in citations: doi:10.1039/D5CB00108K  doi:10.3204/PUBDB-2025-04021

Abstract: Nucleobase-modified nucleoside-5′-triphosphates (NTPs) are important building blocks for the enzymatic synthesis of non-coding RNAs and mRNAs with improved properties. Chemical phosphorylation of base-modified nucleotides to NTPs remains challenging. Here, we report the enzymatic phosphorylation of purine-modified nucleoside-5′-monophosphates (NMPs) to the corresponding NTPs by the polyphosphate kinase 2 class III from an Erysipelotrichaceae bacterium (EbPPK2). The enzyme is highly promiscuous, accepting a range of NMPs with purine modifications. EbPPK2 efficiently catalyses the formation of the corresponding di-, tri- and tetraphosphates, typically with >70% conversion to the NTP. Slower conversion was observed for analogues with oxo- or thio-substitutions at the C6-position. To better understand nucleotide binding and catalysis, we determined the crystal structure of EbPPK2 at 1.7 Å resolution bound to a non-hydrolysable ATP analogue and polyphosphate. This enabled structure-guided design of EbPPK2 variants that efficiently convert GMP analogues, while retaining activity for AMP. Apart from being the preferred industrial-scale ATP recycling catalyst, EbPPK2 and variants bear potential to become the favoured enzyme family for purine-modified NTP production.

Note: A. R. would like to thank DFG for funding (RE2796/10-1 and CRC1459).

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Contributing Institute(s):

1. EMBL-User (EMBL-User)

Research Program(s):

1. 6G3 - PETRA III (DESY) (POF4-6G3) (POF4-6G3)

Experiment(s):

1. PETRA Beamline P13 (PETRA III)

Appears in the scientific report 2025

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