Members of the DUF231 Family are O-Acetyltransferases Catalyzing 2-O- and 3-O-Acetylation of Mannan

Abstract

Mannans are hemicellulosic polysaccharides commonly found in the primary and secondary cell walls of land plants, and their mannosyl residues are often acetylated at O-2 and O-3. Currently, little is known about the genes responsible for the acetylation of mannans. In this report, we investigated the roles of a subgroup of DUF231 proteins including 11 from Arabidopsis thaliana and one from Amorphophallus konjac in mannan acetylation. Acetyltransferase activity assays of their recombinant proteins revealed that four Arabidopsis DUF231 proteins possessed an enzymatic activity capable of transferring acetyl groups from acetyl-CoA onto the mannohexaose acceptor, and thus were named mannan O-acetyltransferases (MOAT1, MOAT2, MOAT3 and MOAT4). Their close homolog from A. konjac (named AkMOAT1) also exhibited mannan O-acetyltransferase activity. Structural analysis of the MOAT-catalyzed reaction products demonstrated that these MOATs catalyzed 2-O- and 3-O-monoacetylation of mannosyl residues, an acetyl substitution pattern similar to that of Arabidopsis glucomannan. Site-directed mutagenesis showed that mutations of the conserved residues in the GDS and DXXH motifs of MOAT3 abolished its acetyltransferase activity, indicating the essential roles of these motifs in its activity. In addition, simultaneous RNA interference (RNAi) inhibition of the expression of the four Arabidopsis MOAT genes led to a drastic reduction in the degree of acetyl substitutions in glucomannan, further corroborating their role in glucomannan acetylation. Together, these results present the first lines of biochemical and genetic evidence demonstrating that these four Arabidopsis DUF231 members and their close A. konjac homolog are mannan O-acetyltransferases.