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Abstract
Xylans are the most abundant hemicellulosic polysaccharides in dicots and are often heavily O-acetylated. The degree of O-acetylation of xylan not only plays a role in maintaining cell wall architecture and plant growth, but it also affects the physicochemical properties and thus our utilization of lignocellulosic biomass. Despite the importance of polysaccharide O-acetylation, the detailed mechanism behind it remained enigmatic until recently. The enzymes involved in O-acetylation of cell wall polysaccharides belong to the TRICHOME BIREFRINGENCE-LIKE (TBL) family, which is composed of 46 members in Arabidopsis thaliana. In this study, my work focused on the biochemical and structural characterization of the most highly expressed TBL protein during secondary wall formation: XYLAN O-ACETYLTRANSFERASE1 (XOAT1). Detailed characterization of XOAT1-catalyzed reactions by real-time NMR showed it regiospecifically 2-O-acetylates xylosyl backbone residues. Structural analysis of XOAT1 revealed a conformation that bears similarities to the ?/β/? topology of the GDSL-like esterase/lipase family and possesses a deep cleft formed by two unequal lobes that accommodate the substrate. Biochemical analyses and molecular dynamics (MD) simulations were used to show that XOAT1 catalyzes acetylation through formation of an acyl-enzyme intermediate by a double displacement bi-bi mechanism involving a Ser-His-Asp catalytic triad and forms an oxyanion hole using an Arg residue. Unfortunately, a ligand-bound structure was unavailable for XOAT1. To gain more insight into the optimal orientation of xylan in the binding cleft of XOAT1 and key residues involved in enzyme-substrate interactions, docking and MD simulations coupled with site-directed mutagenesis and biochemical analyses were used, which revealed the major lobe of XOAT1 is important for xylan binding. The resulting XOAT1 variants with various enzymatic efficiencies were applied to one-pot synthesis of xylan polymers with different degrees of O-acetylation, confirming the utility of this approach. Finally, I also characterized a second Arabidopsis TBL protein, TBL3, and present its novel biochemical activity as a bifunctional xylan 2-O-transacetylase that is able to use acetylated xylan as an acetyl donor, suggesting its role in regulating O-acetylation levels and/or patterning along the xylan backbone. Taken together, these studies provide new molecular level insights into the process of xylan O-acetylation during plant cell wall biosynthesis.