Cholesterol oxidase is a flavoenzyme that catalyzes the oxidation
and isomerization of cholesterol to cholest-4-en-3-one. It is
a water soluble, interfacial enzyme that binds transiently to
the membrane surface during catalysis. Interestingly, it was one
of the early tools used to probe the heterogeneity of cell membranes
and the localization of cholesterol in the lipid bilayer (6).
In addition, cholesterol oxidase has insecticidal properties against
Coeloptera larvae, agricultural pests, (7) and is being developed
for use in agricultural crop treatment. Moreover, some pathogenic
actinomycetes that infect lung macrophages, e.g., Rhodococcus
equi and perhaps Mycobacterium tuberculosis, require cholesterol
oxidase for virulence. We have begun to unravel how bacteria utilize
cholesterol oxidase to alter the physical properties of lipid
membranes. Our kinetic and binding studies have provided insight
into the importance of lipid bilayer structure for substrate specificity.
Our kinetic and structural analyses have elucidated how the chemistry
is catalyzed. Our productive collaboration with Professor Alice
Vrielink at UC Santa Cruz has yielded the ultra-high resolution
X-ray crystal structures of cholesterol oxidase. These structures
have given us insight into the hydrogen bond network in the active
site, and control of redox potential, and the dynamics of the
protein. These studies have prompted additional hypotheses about
the potential use of cholesterol oxidase as a probe of membrane
structure, the structure of the enzyme itself at the lipid bilayer,
and the dynamics of catalytic residues during catalysis.
Our goal is to develop a precise molecular model
of the lipid requirements for activity and of the pathway to flavin
oxidation. These models are important for the development of cholesterol
oxidase as a commercial product, as a useful and reliable tool
in the study of cellular membranes for monitoring lipid rafts,
for investigating the molecular pathway of bacterial pathogenesis,
and for developing anti-bacterial inhibitors.
