The acetylxylan esterase, Axe2, in the thermophilic bacterium Geobacillusstearothermophilus participates in the degradation of the high molecular weight polysaccharide xylan found in the plant cell wall, by removing acetyl groups from the backbones of xylo-oligomers inside the cell. Axe2 represents a new family of carbohydrate esterases belonging to the SGNH superfamily of hydrolytic enzymes with α/β/α fold. Recently the crystal structure of Axe2 was resolved and analyzed, showing a unique doughnut-like octameric configuration composed of four dimeric asymmetric units that form two staggered tetrameric rings. We used site-directed mutagenesis to determine interactions which are responsible for the oligomeric state of the protein, and showed that H-bonds clusters at the interfaces of the dimers were the most significant interactions for stabilizing the octamer, and that the octamer configuration takes part in stabilizing the catalytic loop found at these interfaces. Surprisingly, the uncharacterized homologous protein from Alicyclobacillus acidocaldarius (PDB no. 3RJT), has similar quaternary structure as Axe2 created by un-conserved interfacial interactions. We suggest that the octameric configuration is a favorable common ancestor, which was selected due to change in substrate specificity, substrate binding or protein stability, and that at a later stage various mutations occurred to stabilize the structure leading to the current sequences of the octameric group members.
