Enzymatic formation of a conserved isoaspartate in ribosomal protein uS11

Xue, Y.; Majumdar, C.; Sofela, S. O.; Shulgina, Y.; Nagle, R.; Wu, T.; Mchaourab, H.; Voehler, M. W.; Cate, J. H. D.; Mitchell, D. A.

doi:10.64898/2026.08.26.747336

bioRxiv · 10.64898/2026.08.26.747336

Enzymatic formation of a conserved isoaspartate in ribosomal protein uS11

Xue, Y.·Majumdar, C.·Sofela, S. O.·Shulgina, Y.·Nagle, R.·Wu, T.·Mchaourab, H.·Voehler, M. W.·Cate, J. H. D.·Mitchell, D. A.

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Abstract

Isoaspartate (isoAsp) formation is typically viewed as a "molecular clock" through nonenzymatic degradation of aspartate or asparagine during protein aging. Here we report a nearly universal enzymatic pathway for the formation of a conserved isoAsp in the bacterial ribosomal protein uS11. Proteome-wide protein-protein interaction scans using AlphaFold3 identified YbeY as a candidate enzyme from Escherichia coli. NMR spectroscopy supported a stable YbeY-uS11 complex from Thermotoga maritima. Biochemical assays indicated that EcYbeY catalysis is zinc-dependent and prefers the conserved Asn-Gly motif for isoAsp formation. A high-resolution cryo-electron microscopy structure of the 70S ribosome from E. coli {Delta}ybeY revealed that loss of isoAsp alters contacts with the 16S rRNA groove and bS21. Phylogenetic analysis indicated that YbeY is present in almost all bacteria, and its absence is correlated to changes in the Asn-Gly motif of uS11. Additionally, our structural analyses implicate Fap7 as the functional counterpart in archaea and eukaryotes.

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Xue, Y., Majumdar, C., Sofela, S. O., Shulgina, Y., Nagle, R., Wu, T., Mchaourab, H., Voehler, M. W., Cate, J. H. D., Mitchell, D. A.. 2026-08-27. Enzymatic formation of a conserved isoaspartate in ribosomal protein uS11. https://doi.org/10.64898/2026.08.26.747336

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