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Max Fürst @maxfus.bsky.social · 20/05/2025

Given the enzyme’s reported promiscuity toward NADH, we hypothesised that it tolerates synthetic substrates with similar structure. Courtesy to Jäschke lab developing NAD capture seq, we knew that a commercial enzyme—ADPRC—can generate a click dinucleotide from cheap small molecule alcohols & NAD+

Conversion of NAD+ to a click dinucleotide following the strategy developed for NAD captureSeq
https://www.nature.com/articles/nature14020

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Max Fürst @maxfus.bsky.social · 20/05/2025

Turns out ApbE is happily accepting this substrate, allowing us to label a variety of proteins at N or C terminus, as well as internally, with either alkynyl or azido ribose phosphate moieties, yielding typically full conversion after a few hours of reaction, as determined by MS.

Bioorthogonal handles (alkynyl and azido ADPR) conjugation to various proteins with ADD-tag located C-terminally, N-terminally and at an internal loop. (a) Reaction conditions for the attachment of bioorthogonal handles to target proteins using ApbE. (b) MS analysis showing the successful incorporation of alkynyl and azido ribose phosphate, indicated by mass additions of 281 Da and 296 Da, respectively. "F1" and "F2" denote the tag sequences GVDGLSGATLTS and GVDGLSGATL, respectively.

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Max Fürst @maxfus.bsky.social · 20/05/2025

Because ADPRC works on NAD+, not NADPH, while ApbE only accepts NADPH and ignores NAD+, we can even run the two enzymatic steps (substrate generation & protein modification) in one pot—just add the two enzymes with the precursors NAD+ and the click alkanol to your target protein!

One-pot click chemistry handle attachment (alkynyl-phospho-ribose) to F1-MBP using ADPRC and ApbE in Tris·HCl pH 8 starting from NAD+ and 4-pentyn-1-ol. Complete labelling was observed after 12h reaction.

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