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A rice GT61 glycosyltransferase possesses dual activities mediating 2-O-xylosyl and 2-O-arabinosyl substitutions of xylan

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Abstract

Main conclusion

A member of the rice GT61 clade B is capable of transferring both 2-O-xylosyl and 2-O-arabinosyl residues onto xylan and another member specifically catalyses addition of 2-O-xylosyl residue onto xylan.

Abstract

Grass xylan is substituted predominantly with 3-O-arabinofuranose (Araf) as well as with some minor side chains, such as 2-O-Araf and 2-O-(methyl)glucuronic acid [(Me)GlcA]. 3-O-Arabinosylation of grass xylan has been shown to be catalysed by grass-expanded clade A members of the glycosyltransferase family 61. However, glycosyltransferases mediating 2-O-arabinosylation of grass xylan remain elusive. Here, we performed biochemical studies of two rice GT61 clade B members and found that one of them was capable of transferring both xylosyl (Xyl) and Araf residues from UDP-Xyl and UDP-Araf, respectively, onto xylooligomer acceptors, whereas the other specifically catalysed Xyl transfer onto xylooligomers, indicating that the former is a xylan xylosyl/arabinosyl transferase (named OsXXAT1 herein) and the latter is a xylan xylosyltransferase (named OsXYXT2). Structural analysis of the OsXXAT1- and OsXYXT2-catalysed reaction products revealed that the Xyl and Araf residues were transferred onto O-2 positions of xylooligomers. Furthermore, we demonstrated that OsXXAT1 and OsXYXT2 were able to substitute acetylated xylooligomers, but only OsXXAT1 could xylosylate GlcA-substituted xylooligomers. OsXXAT1 and OsXYXT2 were predicted to adopt a GT-B fold structure and molecular docking revealed candidate amino acid residues at the predicted active site involved in binding of the nucleotide sugar donor and the xylohexaose acceptor substrates. Together, our results establish that OsXXAT1 is a xylan 2-O-xylosyl/2-O-arabinosyl transferase and OsXYXT2 is a xylan 2-O-xylosyltransferase, which expands our knowledge of roles of the GT61 family in grass xylan synthesis.

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Data availability

All data generated during this study are included in this article and its Supplementary Figure files.

Abbreviations

AA:

Anthranilic acid

Araf :

Arabinofuranose

GT:

Glycosyltransferase

MALDI-TOF:

Matrix-assisted laser desorption ionisation time-of-flight

(Me)GlcA:

(Methyl)glucuronic acid

NMR:

Nuclear magnetic resonance

OsXXAT1:

Xylan 2-O-xylosyl/2-O-arabinosyl transferase 1

OsXYXT2:

Xylan 2-O-xylosyltransferase 2

XAT:

Xylan arabinosyltransferase

Xyl:

Xylose

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Acknowledgements

This work was funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences [grant No. DE-FG02-03ER15415].

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Authors and Affiliations

  1. Department of Plant Biology, University of Georgia, Athens, GA, 30602, USA

    Ruiqin Zhong & Zheng-Hua Ye

  2. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, 30602, USA

    Dayong Zhou, Lirong Chen, John P. Rose & Bi-Cheng Wang

  3. Department of Chemistry, University of Georgia, Athens, GA, 30602, USA

    Dennis R. Phillips & Earle R. Adams

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  1. Ruiqin Zhong
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  2. Dayong Zhou
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Contributions

RZ and ZHY conceived and designed research. RZ, DZ, DRP, ERA, LC, JPR, BCW and ZHY conducted experiments and analysed data. RZ, DZ and ZHY wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Zheng-Hua Ye.

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Communicated by De-Yu Xie.

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Zhong, R., Zhou, D., Phillips, D.R. et al. A rice GT61 glycosyltransferase possesses dual activities mediating 2-O-xylosyl and 2-O-arabinosyl substitutions of xylan. Planta 259, 115 (2024). https://doi.org/10.1007/s00425-024-04396-0

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