BibTex format
@article{Liu:2024:10.1021/jacs.4c05955,
author = {Liu, Y and Bineva-Todd, G and Meek, RW and Mazo, L and Piniello, B and Moroz, O and Burnap, SA and Begum, N and Ohara, A and Roustan, C and Tomita, S and Kjaer, S and Polizzi, K and Struwe, WB and Rovira, C and Davies, GJ and Schumann, B},
doi = {10.1021/jacs.4c05955},
journal = {Journal of the American Chemical Society},
pages = {26707--26718},
title = {A bioorthogonal precision tool for human N-acetylglucosaminyltransferase V},
url = {http://dx.doi.org/10.1021/jacs.4c05955},
volume = {146},
year = {2024}
}
RIS format (EndNote, RefMan)
TY - JOUR
AB - Correct elaboration of N-linked glycans in the secretory pathway of human cells is essential in physiology. Early N-glycan biosynthesis follows an assembly line principle before undergoing crucial elaboration points that feature the sequential incorporation of the sugar N-acetylglucosamine (GlcNAc). The activity of GlcNAc transferase V (MGAT5) primes the biosynthesis of an N-glycan antenna that is heavily upregulated in cancer. Still, the functional relevance and substrate choice of MGAT5 are ill-defined. Here, we employ protein engineering to develop a bioorthogonal substrate analog for the activity of MGAT5. Chemoenzymatic synthesis is used to produce a collection of nucleotide-sugar analogs with bulky, bioorthogonal acylamide side chains. We find that WT-MGAT5 displays considerable activity toward such substrate analogues. Protein engineering yields an MGAT5 variant that loses activity against the native nucleotide sugar and increases activity toward a 4-azidobutyramide-containing substrate analogue. By such restriction of substrate specificity, we show that the orthogonal enzyme–substrate pair is suitable to bioorthogonally tag glycoproteins. Through X-ray crystallography and molecular dynamics simulations, we establish the structural basis of MGAT5 engineering, informing the design rules for bioorthogonal precision chemical tools.
AU - Liu,Y
AU - Bineva-Todd,G
AU - Meek,RW
AU - Mazo,L
AU - Piniello,B
AU - Moroz,O
AU - Burnap,SA
AU - Begum,N
AU - Ohara,A
AU - Roustan,C
AU - Tomita,S
AU - Kjaer,S
AU - Polizzi,K
AU - Struwe,WB
AU - Rovira,C
AU - Davies,GJ
AU - Schumann,B
DO - 10.1021/jacs.4c05955
EP - 26718
PY - 2024///
SN - 0002-7863
SP - 26707
TI - A bioorthogonal precision tool for human N-acetylglucosaminyltransferase V
T2 - Journal of the American Chemical Society
UR - http://dx.doi.org/10.1021/jacs.4c05955
VL - 146
ER -