DAAO

D-amino acid oxidase (DAAO, EC 1.4.3.3) is a typical protease that is based on flavin adenine (FDA). The enzyme has high stereoisomeric selectivity and broad spectrum of catalytic substrate, and can be widely used for qualitative and quantitative analysis of D-amino acids, biosensors, production of L-amino acids and α-keto acids. However, the more important value of DAAO is the application of a two-step process for the production of 7-aminocephalosporanic acid (7-ACA). Oxidation of cephalosporin C with D-amino acid oxidase (DAAO) to produce glutaryl-7-aminocephalosporanic acid (GL-7-ACA), followed by deacetylation by GL-7-ACA acylase to produce 7- ACA. In recent years, two-step enzymatic conversion of cephalosporin (CPC) to biocatalyzed production of 7-ACA has attracted increasing attention.

Distribution

DAAO is widely found in liver, brain, and kidney tissues of mammals such as human, rats, rabbits, and pigs. DAAO is also widely present in microorganisms such as algae, Neurospora, Aspergillus, bacteria, Trichoderma morphicum, Saccharomyces cerevisiae, Rhodotorula glutin. Sequence analysis of D-amino acid oxidases from different sources shows that there is a degree of homology between them. The closer the species is, the higher the homology, but there are significant differences in biochemical characteristics.

For example, the binding of mammalian D-amino acid oxidase to the prosthetic FAD is loose, and the prosthetic group is easily lost and makes the DAAO lose its viability. In terms of substrate utilization, the oxidative deaminating activity of cephalosporin C by DAAO from animal sources is relatively low, while DAAO produced by some yeasts has strong binding to FAD and high catalytic activity to CPC side chains.

Physiological Function

Microorganisms, especially yeast, can be grown using D-amino acid oxidases. In addition, D-amino acids also play an important role in the formation of bacterial cell walls. They are constituents of peptidoglycans and muramic acids, but there are few studies on the production of D-amino acids in animals, and most of them are concentrated in vertebrate. Therefore, the physiological role of DAAO in higher animals is not yet clear. D'Aniello et al. believe that in some animals, DAAO and D-aspartate oxidase (DAS-PO) in the body can metabolize exogenous D-amino acids, while acting as an antidote to endogenous D-amino acids. Recent studies have confirmed that D-amino acids play a role in nerve conduction in the brain, and DAAO is responsible for controlling the content of D-amino acids in the brain.

Biological Characteristics

In 1973, pig kidney D-amino acid oxidase (pKDAAO) was the first purified flavoprotein extracted. The complementary DNA (cDNA) of pKDAAO has been completely cloned and sequenced. pKDAAO contains 1041 base pair open reading frame encoding 347 amino acids of the enzyme. Purified D-amino acid oxidase (RgDAAO) was obtained in Rhodotorula chinensis in 1987, which was also the first purification of flavoprotein from microorganisms. The cDNA of RgDAAO was cloned by PCR and the holoenzyme was also performed in E. coli. In 1998, the RgDAAO genome was sequenced, and the entire protein coding region contained 1104 base pairs, and it was confirmed that it contained 5 introns. Then Trigonopsos variabilis D-amino acid oxidase (TvDAAO) was also isolated. The gene was cloned and it was found that the holoenzyme contained 356 amino acids and contained an intron in the genome of TvDAAO.

Catalytic Mechanism

The D-amino acid oxidase oxidizes the amino group of the D-amino acid to form the corresponding keto acid and ammonia. This reaction must be performed with the participation of FAD. The reaction process is divided into three steps:

(1) RCHNH₂COOH + E-FAD → RC=NHCOOH + E-FADH₂
(2) E-FADH₂ + O₂ → E-FAD + H₂O₂
(3) RC=NHCOOH + H₂O → RCOCOOH + NH₃

Reaction (1) is an incomplete reduction reaction, and the D-amino acid deoxygenates into the corresponding imino acid and binds to the reducing FAD. This step is the only enzymatic reaction in the entire reaction. In reaction (2), the reductive FAD is oxidized again in the presence of molecular oxygen and oxygen is converted into hydrogen peroxide. In reaction (3), the imino acid is hydrolyzed to α-keto acid and ammonia without an enzyme. However, when hydrogen peroxide is present in the body, decarboxylation of the α-keto acid occurs to produce the corresponding acid.

Applications

At present, D-amino acid oxidase is mainly used in the two-step enzymatic production of 7-aminocephalosporanic acid. The conversion of CPC to 7-ACA by the two-step enzymatic reaction is as follows: In the first step, D-amino acid oxidase catalyzes the oxidative deamination reaction of D-aminoadipoyl side chain in the CPC molecule under aerobic conditions to generate α-Ketoacid-adipoyl-7-aminocephalosporanic acid (AKA-7-ACA), NH₃ and H₂O₂. The generated H₂O₂ in turn further oxidatively decarboxylates AKA-7-ACA to glutaryl-7-aminocephalosporanic acid (GL-7-ACA). Then, the glutaryl side chain of GL-7-ACA was hydrolyzed by the action of GL-7-ACA acylase to form the final product 7-ACA of the two-step reaction. Using molecular biological methods, the DAAO gene was cloned, expressed and used in industrial production. It is hoped that as a D-amino acid antidote, it can be used in the food industry.