WHAT IS THE PHYSIOLOGICAL FUNCTION OF D-AMINO-ACID OXIDASE?
Ryuichi Konno
Department of Microbiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan
FAX: 0282-86-5616,
E-mail: konno@dokkyomed.ac.jp(Received 26 July 2002 Accepted 30 August 2002)
Abstract
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 mainlypresent 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 wehave such a curious enzyme in our body.
We believed that mutant animals having dysfunctions in DAO would be useful for the elucidation of thephysiological 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 andtheir strain was established.
Genetic cross experiments between the mutant mice and normal mice indicated that mice have one geneencoding DAO and that it is expressed in the kidney and brain. The DAO gene was transmitted through the Mendelianmanner. 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 wasmapped at 65 cM on the chromosome 5 by the linkage analysis. Fluorescence in situ hybridization mapped this geneat 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 ofamino 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 mostimportant 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 wasmainly D-isomer and was determined to originate from the cell walls of intestinal bacteria. Abundant methionine wasalso mostly D-isomer and determined to derive from DL-methionine supplemented in the diet of the mice. Serine wasmostly D-isomer and a portion of the D-serine was indicated to be synthesized in the body. These D-amino acids werenot 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 ofDAO.

Keywords: D-Amino-acid oxidase, physiological function, mutant mice, genetics, point mutation, aminoaciduria, urine

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Fig.1. D- Aminoaciduria in mutanat mice lacking D-amino-acidoxidase. A large amounts of D-methionine, D-methionine
sulfoxide/sulfone, D-alanine, D-serine and D-proline are presentin the urine of mutant mice lacking D-amino-acid oxidase.
Urinary D-methione and D-methionine sulfoxide/sulfone
originate from D-methionine supplemented in the mouse diet.
Urinary D-alanine derives from D-alanine in the cell walls ofintestinal bacteria. A portion of urinary D-serine is synthesizedin the body.

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