Our present experiments are aimed at developing cholesterol oxidase
as a reliable tool for membrane structure studies, identifying
the physiological membrane substrate, and investigating the regulation
of oxygen access to the active site. The specific issues that
we are addressing are outlined below.
1. We hypothesize that cholesterol oxidase can distinguish between
cholesterol-containing lipid domains of different composition
in the same membrane, i.e., between liquid-disordered domains
and liquid-ordered domains (rafts). Current proposals suggest
that removal of the sphingomyelin headgroup to form ceramide forces
sterols out of lipid rafts.34 We are investigating whether inclusion
of sphingomyelin or ceramide in a membrane alters the affinity
of the enzyme for the membrane, the catalytic activity of the
enzyme or both. These experiments will identify what substrate
membrane structure is optimal for catalysis and thus the likely
membrane target of bacteria invading their host macrophage.
2. We hypothesize that the enzyme employs a large interface to
associate with the lipid bilayer during catalysis. We are continuing
to refine our model of protein-lipid binding and are testing how
sensitive binding is to lipid composition. These experiments are
important for developing a model of protein-membrane interaction
that will help identify mammalian membranes targeted by the enzyme.
3. We hypothesize that a gated tunnel between the solvent and
the flavin at the active site controls oxygen access. We are testing
whether this gate and tunnel control oxygen access with a combination
of mutagenesis, kinetic and structural studies. Our mutants are
designed to either sterically block the tunnel or to decouple
the Asn485 gate from tunnel opening. These experiments are important
for understanding how dynamics controls activity at a fundamental
level. Furthermore, they are important for determining the feasibility
of designing inhibitors that target the oxygen tunnel.
4. We have begun studying the Mycobacterium tuberculosis cholesterol
oxidase. These experiments are aimed at determining whether the
function of the putative M. tuberculosis cholesterol oxidase is
correctly assigned to its gene. Next, we will determine its structure
and analyze its pattern of expression in the M. tuberculosis infectious
cycle. The goal of these studies is to determine the role of cholesterol
oxidase in M. tuberculosis pathogenesis.
