Cholesterol oxidase is a bacterial-specific flavoenzyme that catalyzes the oxidation and isomerisation of steroids containing a 3β hydroxyl group and a double bond at the 5–6 of the steroid ring system. The enzyme is a member of a large family of flavin-specific oxidoreductases and is found in two different forms: one where the flavin adenine dinucleotide (FAD) cofactor is covalently linked to the protein and one where the cofactor is non-covalently bound to the protein.
Cholesterol oxidases are secreted bacterial enzymes that catalyze the first step in the degradation of cholesterol. They are flavoenzymes containing the redox cofactor, flavin adenine dinucleotide (FAD). The enzyme catalyzes three chemical conversions. In the first step, called the reductive half-reaction, the 3β-hydroxyl group of the steroid ring system is oxidized to the corresponding ketone. Key to this conversion is the FAD cofactor, which becomes reduced in the process. In the second step the enzyme catalyzes isomerization of the double bond in the oxidized steroid ring system from the 5–6 position to 4–5 position, to give the final steroid product, cholest-4-en-3-one. In the final step of the enzyme, called the oxidative half-reaction, the reduced cofactor reacts with dioxygen and is thus reoxidized while O2 is reduced to H2O2.
Cholesterol oxidase has been used in a study to demonstrate that microheterogeneity in hydrophobic-hydrophilic block copolymer systems can be exploited for immobilizing enzymes and to carry out enzymatic reactions. Cholesterol oxidase has also been used in a study that concluded that amphipaths that activate cholesterol might be useful in treating NPC disease.
Cholesterol oxidase is used to determine serum cholesterol. The enzyme also finds application in the microanalysis of steroids in food samples and in distinguishing 3-ketosteroids from 3b-hydroxysteroids. Transgenic plants expressing cholesterol oxidase are being investigated in the fight against the cotton boll weevil. CHOD has also been used as a molecular probe to elucidate cellular membrane structures.
Cholesterol oxidases are part of a unique class of enzymes that are soluble proteins although they interact with highly insoluble substrates. The natural substrate, cholesterol, is an important membrane component exhibiting low solubility in the aqueous medium of the cell, hence the enzyme must interact with the lipid bilayer in order to for the substrate to partition out of the membrane and undergo oxidation at the enzyme active site. In this regard cholesterol oxidase is an interfacial enzyme as it binds transiently to the membrane surface during catalysis and can only access the substrate from the membrane phase. Other examples of interfacial enzymes include phospholipases (Berg et al., 2001; Gelb et al., 1995; Jain et al., 1995).
Cholesterol oxidase has a number of important commercial applications. Initial studies on the enzyme focused on its use for the determination of cholesterol in serum, HDL or LDL (see review, Smith and Brooks, 1976). Serum cholesterol levels are determined using a three-enzyme assay, including cholesterol esterase, cholesterol oxidase and peroxidase (Richmond, 1973; Allain et al., 1974; Richmond, 1976). More recently electrochemical biosensors with immobilized cholesterol oxidase have been employed to determine cholesterol levels in serum or food (Arya et al., 2007; Basu et al., 2007; Vidal et al., 2004). Determining the serum cholesterol levels is critical for the assessment of a variety of diseases including hypercholesterolemia, coronary heart disease and lipid disorders for estimating the risk of thrombosis and myocardial infarction.
Cholesterol Oxidase produced from Streptomyces sp corresponds to a molecular weight of 62 kDa. It has a pH and temperature optimum of 7 and 37 °C, respectively. Cholesterol Oxidase comprises a FAD-binding and a steroid-binding domain.
Cholesterol oxidase (CHOD) is a monomeric flavoprotein containing FAD that catalyzes the first step in cholesterol catabolism. This bifunctional enzyme oxidizes cholesterol to cholest-5-en-3-one in an FAD-requiring step, which is then isomerized to cholest-4-en-3-one with the release of H2O2.
