High throughput mass spectrometry-based characterisation of Arabidopsis thaliana group H glycosyltransferases

Abstract

In this report, we cloned and characterised four members of group H glycosyltransferases (GTs) by studying their substrate specificities and kinetics. The formation of products and possible glycosylation position was confirmed using MS/MS. The results revealed that 76E1 and 76E5 have broader donor specificity, including UDP-glucose (UDPGlc), UDP-galactose (UDPGal) and UDP-N-acetylglucosamine (UDPGlcNAc) with various flavonoids as acceptor substrates. Pseudo-single substrate kinetics data showed a relatively low K _M, indicating a high affinity for substrate UDPGlc and also supported that 76E5 is more of a galactosyl and N-acetylglucosamine transferase. Sequence alignment and site-directed mutagenesis studies indeed suggested that serine is a crucial residue in the UDPGlcNAc and UDPGal activity.

Fig. 1. MS based method and GAR screening result. (a) The MS/MS method to determine the formation of product using an example of UDPGlcNAc with kaempferol catalyzed by UGT76E5 (inset: reaction scheme and the fragment of product, product structure for illustration purpose only). (b) GAR acceptor screening result of enzymes (c) structures of the compounds showed positive reaction in the acceptor screening.

Fig. 2. Donor screening result. (A) UDPGlc, (B) UDPGal, (C) UDPGlcNAc, (D) GDPFuc, (E) GDPMan, (F) GDPGlc and (G) UDPMan.

Fig. 3. The MS/MS method to determine the formation of product and potentially glycosylation position. (a) An example to show the 3-O glycosylation – the formation of radical aglycone (283.8). (b) An example to show the glycosylation on 4′-O position – the formation of aglycone (284.9) and the presence of fragment of 179.0. (c) An example to show the 7-O glycosylation – the formation of aglycone (300.8) only.

Fig. 4. Sequence alignment of UDPGlcNAc glycosyltransferases and mutant activities. (a) Sequence alignment showing serine thought to be involved in O-galactosylation and O-GlcNAcylation. (b) Sequence alignment showing serine in other Group H UGTs, thought to be involved in O-galactosylation and O-GlcNAcylation, (c) mutants' donor activity results: comparison between wild type enzymes 76E5, 76E1 and 76E2 and mutant enzymes 76E5 S311N, 76E1 S318N and 76E2 N320S activity with donor compounds (A) UDPGlc, (B) UDPGal, (C) UDPGlcNAc. 76E2 N320S mutant newly acquired UDPGal and UDPGlcNAc activities.