Rational Enzyme Design for Controlled Functionalization of Acetylated Xylan for Cell-Free Polymer Biosynthesis

Hsin-Tzu Wang, Vivek Bharadwaj, Jeong-Yeh Yang, Thomas Curry, Kelley Moremen, Yannick Bomble, Breeanna Urbanowicz

Research output: Contribution to journalArticlepeer-review

4 Scopus Citations


Xylan O-acetyltransferase 1 (XOAT1) is involved in O-acetylating the backbone of hemicellulose xylan. Recent structural analysis of XOAT1 showed two unequal lobes forming a cleft that is predicted to accommodate and position xylan acceptors into proximity with the catalytic triad. Here, we used docking and molecular dynamics simulations to investigate the optimal orientation of xylan in the binding cleft of XOAT1 and identify putative key residues (Gln445 and Arg444 on Minor lobe & Asn312, Met311 and Asp403 on Major lobe) involved in substrate interactions. Site-directed mutagenesis coupled with biochemical analyses revealed the major lobe of XOAT1 is important for xylan binding. Mutation of single key residues yielded XOAT1 variants with various enzymatic efficiencies that are applicable to one-pot synthesis of xylan polymers with different degrees of O-acetylation. Taken together, our results demonstrate the effectiveness of computational modeling in guiding enzyme engineering aimed at modulating xylan and redesigning plant cell walls.

Original languageAmerican English
Article number118564
Number of pages10
JournalCarbohydrate Polymers
StatePublished - 2021

Bibliographical note

Publisher Copyright:
© 2021

NREL Publication Number

  • NREL/JA-2800-80962


  • Computational modeling
  • Enzyme engineering
  • Enzyme-substrate interaction
  • One-pot synthesis
  • Xylan O-acetylation
  • Xylooligosaccharide


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