Molecular Mechanism of Polysaccharide Acetylation by the Arabidopsis Xylan O-acetyltransferase XOAT1

• Published in: The Plant cell Vol. 32; no. 7; pp. 2367 - 2382
• Main Authors: Lunin, Vladimir V., Wang, Hsin-Tzu, Bharadwaj, Vivek S., Alahuhta, Markus, Peña, Maria J., Yang, Jeong-Yeh, Archer-Hartmann, Stephanie A., Azadi, Parastoo, Himmel, Michael E., Moremen, Kelley W., York, William S., Bomble, Yannick J., Urbanowicz, Breeanna R.
• Format: Journal Article
• Language: English
• Published: England American Society of Plant Biologists 01.07.2020; Oxford University Press
• Subjects: Acetylation; Acetyltransferases - chemistry; Acetyltransferases - genetics; Acetyltransferases - metabolism; Arabidopsis - metabolism; Arabidopsis Proteins - chemistry; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Arginine - metabolism; Catalysis; Catalytic Domain; Crystallography, X-Ray; HEK293 Cells; Humans; Magnetic Resonance Spectroscopy; Models, Molecular; Mutation; Polysaccharides - metabolism; Protein Conformation; Xylans - metabolism
• ISSN: 1040-4651; 1532-298X; 1532-298X
• DOI: 10.1105/tpc.20.00028

Structural and biochemical analysis of xylan O-acetyltransferase 1 (XOAT1) show that XOAT1 catalyzes xylan acetylation through formation of an acyl-enzyme intermediate via a double displacement bi-bi mechanism. Abstract Xylans are a major component of plant cell walls. O-Acetyl moieties are the dominant backbone substituents of glucuronoxylan in dicots and play a major role in the polymer-polymer interactions that are crucial for wall architecture and normal plant development. Here, we describe the biochemical, structural, and mechanistic characterization of Arabidopsis (Arabidopsis thaliana) xylan O-acetyltransferase 1 (XOAT1), a member of the plant-specific Trichome Birefringence Like (TBL) family. Detailed characterization of XOAT1-catalyzed reactions by real-time NMR confirms that it exclusively catalyzes the 2-O-acetylation of xylan, followed by nonenzymatic acetyl migration to the O-3 position, resulting in products that are monoacetylated at both O-2 and O-3 positions. In addition, we report the crystal structure of the catalytic domain of XOAT1, which adopts a unique conformation that bears some similarities to the α/β/α topology of members of the GDSL-like lipase/acylhydrolase family. Finally, we use a combination of biochemical analyses, mutagenesis, and molecular simulations to show that XOAT1 catalyzes xylan acetylation through formation of an acyl-enzyme intermediate, Ac–Ser-216, by a double displacement bi-bi mechanism involving a Ser-His-Asp catalytic triad and unconventionally uses an Arg residue in the formation of an oxyanion hole.