Structural characterization of the organic solvent-stable cholesterol oxidase from Chromobacterium sp. DS-1.
M.Sagermann,
A.Ohtaki,
K.Newton,
N.Doukyu.
ABSTRACT
Cholesterol oxidase is of significant commercial interest as it is widely used
as a biosensor for the detection of cholesterol in clinical samples, blood serum
and food. Increased stability of this enzyme with regards to temperature and
different solvent conditions are of great importance to the reliability and
versatility of its applications. We here report the crystal structure of the
cholesterol oxidase of Chromobacterium sp. DS-1 (CHOLOX). In contrast to other
previously characterized cholesterol oxidases, this enzyme retains high activity
in organic solvents and detergents at temperatures above 85 degrees C despite
its mesophilic origin. With the availability of one other homologous oxidase of
known three-dimensional structure, a detailed comparison of its sequence and
structure was performed to elucidate the mechanisms of stabilization. In
contrast to factors that typically contribute to the stability of thermophilic
proteins, the structure of CHOLOX exhibits a larger overall cavity volume, less
charged residues and less salt bridge interactions. Moreover, the vast majority
of residue substitutions were found on or near the protein's solvent exposed
surface. We propose that the engineering of enhanced stability may also be
accomplished through selective engineering of the protein periphery rather than
by redesigning its entire core.
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