(618a) Tertiary Structure and Properties of a Glycoside Hydrolase Family 44 Endoglucanase from Clostridium Acetobutylicum

CAC0915, an
endoglucanase whose genetic coding exists in Clostridium acetobutylicum
but which is not normally expressed by it, was expressed by Escherichia coli
after is gene was synthesized. It belongs to glycoside hydrolase family 44 and
has been structurally and kinetically characterized. The crystal structure of
CAC0915 was solved to 2.2 Å resolution, revealing a TIM barrel structure with a
Greek key fold and b-sandwich domain. The catalytic acid,
Glu180, and base, Glu352, are located 5.5 Å apart on the fourth and seventh
beta strands of the TIM barrel. The catalytic acid is 4.5 Å from the glycosidic
oxygen suggesting a rearrangement occurs upon ligand binding. This
rearrangement maybe facilitated by a calcium atom, located behind the catalytic
acid, which forms a kink in the peptide backbone. A catalytic triad composed of
Glu180-His277-Ser351 raises the pK_a of the catalytic acid.

Substrates bind
in a cleft composed of nine subsites, ?4 through +5. Subsites ?4 through   ?1
contain three aromatic residues (Trp58, Tyr65, and Trp385) which form stacking
interactions with the substrate. Four hydrogen bonds are also present in the
minus subsites, two of which help distort the pyranose ring in subsite ?1.
Subsites +1 to +5 contain one hydrogen bond, in subsite +2, and two aromatic
residues (Trp320 and Trp324) in subsites +4 and +5. The topography of the
binding cleft suggests the substrate will have higher affinity for subsites ?1
to ?4 than for subsites +1 to +5. This is consistent with the results of
reaction product analysis on cellopentaose and cellohexaose substrates using
thin layer chromatography. Cellopentaose produces cellotetraose and glucose and
cellohexaose produces cellotriose and cellobiose, respectively, as primary
products. However, both substrates also produce secondary reaction products,
showing the substrate can bind in subsites ?3 to +2 or ?3 to +3.

CAC0915
hydrolyzes carboxylmethyl cellulose, birchwood xylan, larchwood xylan,
cellopentaose, and cellohexaose. It is not active on mannan, laminarin, Avicel,
pullulan, or lichenan. Michaelis?Menten kinetic parameters were determined on
carboxymethyl cellulose (k_cat18.9 U/mg, K_M
0.263 g/L), birchwood xylan (k_cat31.6 U/mg, K_M
0.412 g/L) and larchwood xylan (k_cat29.5 U/mg, K_M
0.278 g/L). CAC0915 is optimally active at pH 5.0 and has an activation energy
for activity of 31.3 ± 2.3 kJ/mol-K and an activation energy for inactivation
of 227 ± 20.5 kJ/mol-K.