Author

Dmitri V. Filippov

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Open access Jul 2026

Identification of cellular intermediates unveils unique enzymes for flagellar glycan biosynthesis in Clostridioides difficile.

Glycosylation of bacterial surface proteins, such as flagellin (FliC), is important for their function and is often involved in virulence of pathogens. Glycans can be further modified by so-called postglycosylation modifications (PGMs), often resulting in exclusive molecular structures. In Clostridioides difficile, a unique glycan structure (Type A) decorates FliC (which forms the flagellar filament) that consists of an O-linked N-acetyl-β-d-glucosamine (GlcNAc) modified with an N-methyl-L-threonine via a phosphodiester linkage. This PGM is synthesized by a set of four enzymes encoded in one operon (ftaABCD), but the exact biosynthesis pathway and biosynthetic intermediates remain unknown. In this study, we chemically synthesized two hitherto undescribed biosynthetic intermediates that we predicted based on bioinformatic analyses, CDP-threonine and CDP-N-methylthreonine. We showed that they are involved in the Type A PGM biosynthesis, as evidenced by mass spectrometric analyses of extracts of a set of C. difficile mutant strains. Furthermore, we characterized FtaC to be a SAM-dependent CDP-threonine N-methyltransferase that installs the methyl group on CDP-threonine prior to transfer of the PGM to GlcNAc-FliC, and we revealed FtaD as the CDP-N-methylthreonine:GlcNAc N-methylthreoninephosphotransferase. Finally, using recombinantly expressed FtaC and FtaD in combination with synthetic CDP-threonine, we reconstituted the biosynthesis pathway of the Type A PGM in vitro. Overall, our results open avenues to explore these unique biosynthesis enzymes in molecular detail to provide new points of entry for the development of biosynthesis inhibitors and tools to study the role of this PGM in virulence and flagellar function.

P. Hensbergen, Bob van Puffelen, Nina Musch et al. · Proceedings of the National... · 0 citations

Jul 2026

Deciphering the Specificity of Reversible DNA-Phosphate ADP-Ribosylation via the Precise Synthesis and Enzymatic Profiling of Nucleotide-Phospho-ADP-Ribosyl Probes.

ADP-ribosylation (ADPr), long recognized as a canonical protein post-translational modification, has recently expanded to include targeting nucleic acids, uncovering a diverse landscape of noncanonical biological functions. Emerging evidence suggests that ADPr at the 5'-phosphate terminus of DNA is implicated in the DNA damage response, yet understanding its precise molecular function has been hampered by the lack of structurally defined chemical probes. Here, we report the stereoselective synthesis of deoxynucleotide-phospho-ADPr (dN-P-ADPr) probes, representing native fragments of terminal DNA-ADPr. Our strategy leverages a mild, stereocontrolled glycosylation to construct the challenging ribosyl-phosphate linkage, followed by P(III)-P(V) coupling to establish the pyrophosphate bridge. This robust toolkit enabled the systematic biochemical profiling of DNA-ADPr hydrolases across diverse kingdoms of life. Remarkably, using these newly developed probes, we uncover hydrolases across the diversity of life capable of reversing ADPr modifications at phosphorylated DNA ends. We further show that these enzymes exhibit an absolute preference for the native-like α-anomer, independent of the identity of the adjacent DNA nucleobase, suggesting that substrate recognition is governed primarily by the ADPr-phosphate linkages rather than the local nucleobase context. Together, these synthetic probes and biochemical insights provide an essential foundation for deciphering the biological landscape of noncanonical ADPr.

Lingxiao Liu, Yang Lu, Zongxing Yu et al. · Journal of the American Chem... · 0 citations