9IGR

Crystal structure of PPK2 class III from Erysipelotrichaceae bacterium in complex with polyphosphate

Summary for 9IGR

• Entry DOI: 10.2210/pdb9igr/pdb
• Related: 9IGQ
• Descriptor: Polyphosphate--nucleotide phosphotransferase, bis[oxidanyl-[oxidanyl-[oxidanyl-[oxidanyl(phosphonooxy)phosphoryl]oxy-phosphoryl]oxy-phosphoryl]oxy-phosphoryl] hydrogen phosphate, BENZOIC ACID, ... (6 entities in total)
• Functional Keywords: polyphosphate kinase, ppk2-iii, ntp transferase, transferase
• Biological source: Erysipelotrichaceae bacterium
• Total number of polymer chains: 4
• Total formula weight: 150669.92

Authors

Rasche, R.,Lawrence-Doerner, A.-M.,Cornelissen, N.V.,Kuemmel, D. (deposition date: 2025-02-20, release date: 2025-07-23, Last modification date: 2025-08-13)

Primary citation

Mitton-Fry, R.M.,Rasche, R.,Lawrence-Dorner, A.M.,Eschenbach, J.,Tekath, A.,Rentmeister, A.,Kummel, D.,Cornelissen, N.V.
Structure-guided engineering of a polyphosphate kinase 2 class III from an Erysipelotrichaceae bacterium to produce base-modified purine nucleotides.
Rsc Chem Biol, 6:1328-1335, 2025

PubMed 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 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.

PubMed: 40667417
DOI: 10.1039/d5cb00108k

Experimental method

X-RAY DIFFRACTION (2.311 Å)