CHOD

Cholesterol oxidase (3β-hydroxysterol oxidase, ChOx, EC1.1.3.6) was first discovered in 1943 and belongs to the flavoprotein oxidoreductase. Cholesterol oxidase mainly catalyzes the dehydrogenation of the C-3 of cholesterol. In addition, it is an isomerization enzyme that catalyzes the isomerization of cholester-5-en-3-one to cholest-4-en-3-one after dehydrogenation of cholesterol. Thus, cholesterol oxidase is a bifunctional enzyme that catalyzes cholesterol to produce the final products cholest-4-en-3-one and hydrogen peroxide.

Classification

The flavin prosthetic group of cholesterol oxidase includes FAD (flavin adenine dinucleotide) and FMN (riboflavin monophosphate), and the prosthetic group of most of the enzymes is FAD, and only the prosthetic group of one enzyme is currently found to be FMN. Cholesterol oxidase can be divided into two main categories. In the class I cholesterol oxidase, FAD and the enzyme are non-covalently bonded. This class of enzymes belong to glucose-methanol-choline oxidoreductases, including cholesterol oxidases derived from Streptomyces sp, B. sterolicum, Rhodococcus equi, and Mycobacterium spp. According to the close non-covalent binding properties of such enzymes and FAD, small molecule ligands can be designed to purify the enzyme in one step. The purification method of the nucleophilic chromatography can finally achieve a protein purity of 95%. In the class II cholesterol oxidase, FAD is covalently bound to the enzyme. FAD is covalently linked to the ND atom or NE atom of the imidazole ring on the histidine residue of the enzyme protein through the methyl group on the isoxazine ring. Representative of class II cholesterol oxidases are from R. erythropolis, Burkholderia spp. and Chromobacterium sp., which are also known as vanillyl alcohol oxidase (VAO). Some strains, such as B. sterolicum, can produce two classes of cholesterol oxidases simultaneously.

Sources

Cholesterol oxidase is generally derived from bacteria and actinomycetes, which are produced by both Gram-negative and Gram-positive bacteria. In addition to the enzyme-producing microorganisms mentioned above, many new strains producing cholesterol oxidase have been discovered in recent years, including B. subtilis, Enterococcus hirae, Pseudomonas aeruginosa, Chryseobacterium gleum. In addition, the presence of cholesterol oxidase has also been found in philophilic microorganisms. Of course, these cholesterol oxidases also have a preference for properties. For example, the optimum pH of an enzyme derived from an alkalophilic microorganism is 10, and the optimum pH of a neutral cholesterol oxidase is generally about 7.

Structure

Cholesterol oxidase generally consists of two domains, a prosthetic binding domain and a substrate binding domain. In the class I cholesterol oxidase, the characteristic sequence of GXGXGGA18XE is generally present in the prosthetic binding domain; the corresponding characteristic sequence in the class II cholesterol oxidase is GXGXXG19XD/E. The structure of the catalytic center of the two classes of enzymes is quite different. For the class I cholesterol oxidase, the catalytic center is generally composed of His, Glu, Asn; and the corresponding residue in the class II cholesterol oxidase is Arg, Glu, Asn.

Catalytic Mechanism

The catalytic process of cholesterol oxidase is mainly divided into the following two steps:

(1) Dehydrogenation: The hydroxyl group at the C-3 of cholesterol carbon is catalytically oxidized by cholesterol oxidase to form cholest-5-en-3-one. In this process, the two oxidized flavin molecules are reduced by two electrons transferred out by catalytic reaction. The reduced flavin molecule reacts with oxygen to form hydrogen peroxide and returns to the oxidized state. The reaction at this stage follows a heterogeneous cleavage (hydride transfer) mechanism.

(2) Isomerization: The oxidized intermediate cholest-5-en-3-one is isomerized under the catalysis of cholesterol oxidase to form cholest-5-en-3-one.

Figure 1. Mechanism of reaction catalyzed by cholesterol oxidase. (Kumari L. 2012)

The dehydrogenation reaction is the rate limiting step of this catalytic process.

Applications

• Detecting serum cholesterol levels

The current method for determining human serum cholesterol is an enzyme coupling method. In recent years, due to the rise of bionics and materials science, the application of this method in the field of medical detection has been further strengthened. For example, cholesterol oxidase can be made into a cholesterol biosensor by fixing it on the surface of the material. The application of the biosensor can effectively improve the utilization efficiency of the enzyme and greatly reduce the cost of the detection method.

• Pest control

Cholesterol oxidase can effectively kill the cotton bollworm larvae, and the biological activity is comparable to the insecticidal protein activity of Bacillus thuringiensis. Cholesterol oxidase can also be applied to the killing of some lepidopteran larvae. The insecticidal mechanism indicates that cholesterol oxidase causes the death of the worm by lysing the epithelial cells of the larval midgut.

• Antibiotic target

Rhodococcus equi is a pathogenic microorganism that can infect young horses. In addition, it is also a conditional pathogen for humans. This microorganism invades the host cell by destroying the cell membrane structure mainly by cholesterol oxidase, thereby invading the host cell, and is extremely harmful to the immunodeficiency patient. Cholesterol oxidase also plays an important role in the infection of human individuals by Mycobacterium tuberculosis, another pathogen that poses a serious threat to humans. In this process, the cholesterol oxidase of the pathogen first weakens the host cell defense system by binding to antibodies on the surface of macrophages. Therefore, cholesterol oxidase can be used as a target for screening new antibiotics. The advantage of choosing this target is that cholesterol oxidase is absent in mammalian cells, so the probability of accidental injury to animal cells by the selected antibiotics is very low.

Reference