D-Amino acid oxidase (DAO) catalyzes oxidative deamination of D-amino acids (steroisomers of naturally occurring L-amino acids), producing 2-oxo acids, hydrogen peroxide and ammonia. In higher animals DAO is mainly present in the kidney, liver and brain. However, since its substrates (D-amino acids) are considered very rare in higher animals, the physiological role of DAO has been enigmatic since its discovery in 1935. We are interested in why we have such a curious enzyme in our body.
We believed that mutant animals having dysfunctions in DAO would be useful for the elucidation of the physiological function of this enzyme. We chose mice as our experimental animals due to their small size, short generation time, clear genetic background, and their close relationship to humans. A screening method for mutant mice possessing reduced levels of DAO activity was devised. Mutant mice lacking DAO activity were isolated and their strain was established.
Genetic cross experiments between the mutant mice and normal mice indicated that mice have one gene encoding DAO and that it is expressed in the kidney and brain. The DAO gene was transmitted through the Mendelian manner. The cross experiments revealed a gene dosage effect on DAO activity: mice carrying two normal DAO alleles have twice the DAO activity than mice carrying one normal and one mutant DAO allele. The DAO gene was mapped at 65 cM on the chromosome 5 by the linkage analysis. Fluorescence in situ hybridization mapped this gene at E3-F on chromosome 5.
The nucleotide sequence analysis of the cloned DAO cDNA revealed the presence of a nucleotide substitution in the middle of the coding region of mutant DAO. This nucleotide substitution caused a substitution of amino acid residue of DAO. The transfection and expression of the mutant and normalized DAO cDNA in cells in culture verified that the nucleotide substitution was the real cause of the loss of DAO activity.
From the nutritional experiments using the mutant and normal mice, DAO was shown to be the most important and indispensable enzyme in the utilization of D-amino acids in the body.
Large amounts of amino acids were detected in the urine of the mutant mice. Most abundant alanine was mainly D-isomer and was determined to originate from the cell walls of intestinal bacteria. Abundant methionine was also mostly D-isomer and determined to derive from DL-methionine supplemented in the diet of the mice. Serine was mostly D-isomer and a portion of the D-serine was indicated to be synthesized in the body. These D-amino acids were not observed in the urine of normal mice. That means that they are constantly metabolized by DAO in normal mice. Therefore, we conclude that the metabolism of endogenous and exogenous D-amino acids is the physiological role of DAO.