jp408767j_si_002.pdf (1.09 MB)
Experimental and Modeling Studies of an Unusual Water-Filled Pore Structure with Possible Mechanistic Implications in Family 48 Cellulases
journal contribution
posted on 2014-03-06, 00:00 authored by Mo Chen, Maxim Kostylev, Yannick
J. Bomble, Michael F. Crowley, Michael E. Himmel, David B. Wilson, John W. BradyMolecular dynamics simulations were
used to study the possible
catalytic role of an unusual conserved water-filled pore structure
in the family 48 cellulase enzyme Cel48A from Thermobifida
fusca. It was hypothesized that this pore serves as
the pathway for the water molecules consumed in the hydrolysis catalyzed
by the enzyme to reach the active site in a continuous stream to participate
in the processive reactions. Theoretical mutants of this enzyme were
created in which all of the residues lining the pore were made hydrophobic,
which had the effect in molecular dynamics simulations of emptying
the pore of water molecules and preventing any from passing through
the pore on the simulation time scale. Mutants with smaller numbers
of substitutions of this nature, which could be created experimentally
by site-directed mutagenesis, were also identified from simulations,
and these proteins were subsequently produced in Escherichia
coli, expressed and purified, but were found to not
fold in a manner similar to the wild type protein, preventing the
determination of the importance of the water pore for activity. It
is possible that the presence of a small vacuum in the pore was responsible
for the instability of the mutants. In addition, alternate pathways
were observed in the simulations that would allow water molecules
to reach the active site of the enzyme, suggesting that the hypothesis
that the pore has functional significance might be incorrect.