D-Amino Acid Oxidase from porcine kidney - ≥2 units/mg dry weight, high purity , CAS No.9000-88-8

Item Number

A128539

• Synonyms: DAO;D-AAO;D-Amino acid;oxygen oxidoreductase(deaminating)
• Specifications & Purity: EnzymoPure™, ≥2 units/mg dry weight
• Storage Temp: Store at 2-8°C
• Shipped In: Wet ice
• Grade: EnzymoPure™

Product Description

Specificity:

The D-isomers of proline, methionine, isoleucine, alanine, valine and phenylalanine are good substrates (Scannone et al. 1964_z and Dixon and Kleppe 1965b). The enzyme is reported to act on L-proline (Wellner and Scannone 1964) and D-lactate (Yagi and Ozawa 1964b). The best substrate for pkDAOO is D-proline, and DAAOs exhibit very poor or no activity toward D-aspartate (Tishkov and Khoronenkova 2005).

The substrate-binding domains in various species' primary structures do not show high homology. This may reflect the wide variation in specificities observed for DAAOs from different origins (Tishkov and Khoronenkova 2005).

Composition:

The active pkDAAO holoenzyme is a monomer of 347 amino acids that can undergo dimerization. The monomer has been found to be more active than the dimer; and contains 1 mol FAD noncovalently bound per monomer. All DAAOs characterized as of 2000 contain noncovalently bound FAD as their prosthetic group (Pilone 2000).

Molecular Characteristics:

The gene encoding mammalian DAAO is present in a single copy in the genome. A 1041 bp open reading frame encodes all 347 amino acids of the enzyme. This indicates posttranslational processing by proteolytic enzymes does not occur (Fukui 1987).

The primary structure of porcine D-amino acid oxidase was determined by Ronchi et aL (Ronchi et al. 1982), and the gene was cloned by Momoi et al. (Momoi et al. 1988). There are six regions of the primary structure that are highly conserved in DAAOs of various sources (Faotto et al. 1995). Regions I contains the consensus sequence GXGXXG, and both regions I and III have been found to be involved in coenzyme binding (Wierenga et al. 1983). Regions II, IV, and V contain the active site residues. The Ser-Lys/His-Leu terminal sequence is the peroxisomal targeting signal sequence (Subramani 1993, and Pilone 2000)

Mammalian DAAOs show 63% identity, and the three known DAAOs of microorganisms (R. gracilis, T. variabilis, and Fusarium solanii') show a 18% identity. 30% identity is observed between yeast and mammalian DAAOs (Pilone 2000).

Protein Accession Number: P00371

CATH Classification (v. 3.2.0):

• Class: Alpha Beta

• Architecture: 2-Layer Sandwich and 3-Layer (aba) Sandwich

• Topology: D-Amino Acid Oxidase; Chain A, domain 2 and Rossmann fold

Molecular Weight:

• 78.7 kDa (Theoretical)

• Monomeric: 38.0-39.0 kDa (Curti et al. 1973, and Tu et al. 1973)

Optimal pH: Dependent on the substrate: approximately 9 for D-alanine (Dixon and Kleppe 1965c).

Isoelectric Point: 7.0_z 7.2 (Tishkov and Khoronenkova 2005)

Extinction Coefficient:

• 75,420 cm'¹ M'¹ (Theoretical)

• Ei%,280 = 19.17 (Theoretical)

Active Site Residues:

• Tyrosine (Y224)

• Aspartic acid (D228)

• Arginine (R283)

(Pilone 2000)

Inhibitors:

• 2-hydroxy acids, 2-oxo acids, and 2-oxobutyrate (Dixon 1965b)

• Metabolites and drugs (Hamilton and Buckthal 1982)

• Adenosine 5^z-monophosphate and aniline (Yagi et al. 1972c)

• Benzoate (Pollegioni et al. 2007)

• Sodium benzoate (Nguyen et al. 2009)

Applications:

• Keto acid preparation

• Oxidation reduction studies

• Separation of L-amino acids from racemic mixtures

• FAD determination

• D-alanine determination

• Biosensors (Inaba et al. 2003)