Harnessing the acceptor substrate promiscuity of Clostridium botulinum Maf glycosyltransferase to glyco-engineer mini-flagellin protein chimeras.
Substrate Specificity
Glycosyltransferases
/ metabolism
Flagellin
/ metabolism
Clostridium botulinum
/ enzymology
Glycosylation
Escherichia coli
/ genetics
Sugar Acids
/ metabolism
Protein Engineering
N-Acetylneuraminic Acid
/ metabolism
Recombinant Fusion Proteins
/ metabolism
Bacterial Proteins
/ metabolism
Sialic Acids
Journal
Communications biology
ISSN: 2399-3642
Titre abrégé: Commun Biol
Pays: England
ID NLM: 101719179
Informations de publication
Date de publication:
21 Aug 2024
21 Aug 2024
Historique:
received:
14
03
2024
accepted:
14
08
2024
medline:
22
8
2024
pubmed:
22
8
2024
entrez:
21
8
2024
Statut:
epublish
Résumé
Several bacterial flagellins are O-glycosylated with nonulosonic acids on surface-exposed Serine/Threonine residues by Maf glycosyltransferases. The Clostridium botulinum Maf glycosyltransferase (CbMaf) displays considerable donor substrate promiscuity, enabling flagellin O-glycosylation with N-acetyl neuraminic acid (Neu5Ac) and 3-deoxy-D-manno-octulosonic acid in the absence of the native nonulosonic acid, a legionaminic acid derivative. Here, we have explored the sequence/structure attributes of the acceptor substrate, flagellin, required by CbMaf glycosyltransferase for glycosylation with Neu5Ac and KDO, by co-expressing C. botulinum flagellin constructs with CbMaf glycosyltransferase in an E. coli strain producing cytidine-5'-monophosphate (CMP)-activated Neu5Ac, and employing intact mass spectrometry analysis and sialic acid-specific flagellin biotinylation as readouts. We found that CbMaf was able to glycosylate mini-flagellin constructs containing shortened alpha-helical secondary structural scaffolds and reduced surface-accessible loop regions, but not non-cognate flagellin. Our experiments indicated that CbMaf glycosyltransferase recognizes individual Ser/Thr residues in their local surface-accessible conformations, in turn, supported in place by the secondary structural scaffold. Further, CbMaf glycosyltransferase also robustly glycosylated chimeric proteins constructed by grafting cognate mini-flagellin sequences onto an unrelated beta-sandwich protein. Our recombinant engineering experiments highlight the potential of CbMaf glycosyltransferase in future glycoengineering applications, especially for the neo-O-sialylation of proteins, employing E. coli strains expressing CMP-Neu5Ac (and not CMP-KDO).
Identifiants
pubmed: 39169227
doi: 10.1038/s42003-024-06736-y
pii: 10.1038/s42003-024-06736-y
doi:
Substances chimiques
Glycosyltransferases
EC 2.4.-
Flagellin
12777-81-0
Sugar Acids
0
N-Acetylneuraminic Acid
GZP2782OP0
2-keto-3-deoxyoctonate
1069-03-0
Recombinant Fusion Proteins
0
Bacterial Proteins
0
5,7-diacetamido-8-O-acetyl-3,5,7,9-tetradeoxy-glycero-talo-nonulosonic acid
0
Sialic Acids
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1029Informations de copyright
© 2024. The Author(s).
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