The
thermophilic soil bacterium Geobacillus stearothermophilus T-6 produces
an intracellular acetylxylan esterase, Axe2, which removes acetyl groups from
xylo-oligosaccharides. The enzyme is a GDSL hydrolase, using the catalytic
triad Ser-His-Asp for hydrolysis, and presents a new undefined family of
carbohydrate esterases in the CAZy database. Axe2 was biochemically
characterized and its regioselectivity was determined on xylopyranoside per
acetate (Alalouf et al. JBC 286:41993, 2011). The crystal
structures of sel-Met Axe2 and its catalytic mutant, S15A, were determined at
1.7 and 1.9 Å resolutions,
respectively, and contain a dimer in the asymmetric unit cell. Conserved residues associated with the
oxyanion hole in the GDSL hydrolase family are positioned at distances
appropriate for hydrogen bonds stabilizing the tetrahedral intermediate in the
reaction mechanism. A single phosphate
anion was found in the active site, simulating the position of acetyl group in
the oxyanion hole during catalysis. Native Axe2 appears to have a Mw of 200,000
based on gel filtration chromatography suggesting that the enzyme is an octamer.
Transmission electron microscopy (TEM) images of the native protein revealed doughnut-like
shape structures of 8-10 nm in diameter in agreement with the crystal
structure. Several conserved residues that form salt bridges, hydrogen bonds
and stacking interactions appear to maintain the octameric structure. The
replacement of Y184F resulted in a dimer structure and suggested that a single
hydroxyl group is crucial for maintaining the native octameric configuration of
Axe2. The W190I replacement resulted in lower activity and demonstrates the
importance of the octameric configuration in stabilizing a loop in the active
site. Additional residues are now being replaced to verify their exact role in
the formation of the octamer and their contribution to the activity and thermal
stability of the protein.
