D-Amino acid oxidase: new findings

Abstract

The most recent research on D-amino acid oxidases and D-amino acid metabolism has revealed new, intriguing properties of the flavoenzyme and enlighted novel biotechnological uses of this catalyst. Concerning the in vivo function of the enzyme, new findings on the physiological role of D-amino acid oxidase point to a detoxifying function of the enzyme in metabolizing exogenous D-amino acids in animals. A novel role in modulating the level of D-serine in brain has also been proposed for the enzyme. At the molecular level, site-directed mutagenesis studies on the pig kidney D-amino acid oxidase and, more recently, on the enzyme from the yeast Rhodotorula gracilis indicated that the few conserved residues of the active site do not play a role in acid-base catalysis but rather are involved in substrate interactions. The three-dimensional structure of the enzyme was recently determined from two different sources: at 2.5-3.0 A resolution for DAAO from pig kidney and at 1.2-1.8 A resolution for R. gracilis. The active site can be clearly depicted: the striking absence of essential residues acting in acid-base catalysis and the mode of substrate orientation into the active site, taken together with the results of free-energy correlation studies, clearly support a hydrid transfer type of mechanism in which the orbital steering between the substrate and the isoalloxazine atoms plays a crucial role during catalysis.

Unfolding Intermediate in the Peroxisomal Flavoprotein d-Amino Acid Oxidase

“…The ⌬ loop mutant DAAO shows slightly altered spectral and kinetic properties, a lower temperature stability, and a 5-fold increase in the K d for FAD binding compared with the wild-type enzyme. We also demonstrated the possibility of obtaining a monomeric form of yeast DAAO by treatment with 0.5 M NH 4 SCN, without deletion of the ␤F5-␤F6 loop (14), as well as by removal of the coenzyme to yield the corresponding apoprotein (10). This latter result suggests a structural relationship between the FAD-harboring domain and the regions involved in dimerization.…”

“…Starting from a 10-liter fermentation broth, 180 and 80 mg of pure enzyme with a specific activity of 110 and 86 units/mg protein were obtained for wild-type and ⌬ loop DAAO, respectively. The enzyme concentration was determined by using extinction coefficients at 455 nm of 12.6 mM Ϫ1 cm Ϫ1 for wild-type and 11.3 mM Ϫ1 cm Ϫ1 for ⌬ loop DAAOs (10,13). Urea was from Pierce, and the other reagents were of analytical grade.…”

“…DAAOs have been the object of extensive investigation (9,10). In solution, DAAO from R. gracilis is a stable 80-kDa homodimer, with a molecule of FAD tightly (K d ϭ 2 ϫ 10 Ϫ8 M) but noncovalently bound to each 40-kDa subunit.…”

Novel insights into renal d-amino acid oxidase accumulation: propiverine changes DAAO localization and peroxisomal size in vivo

“…Subsequently, the accumulated protein was isolated and characterized thereby suggesting the predominant accumulation of the protein D-amino acid oxidase (DAAO, EC 1.4.3.3) (Dietrich et al 2008). DAAO is a FAD-dependent peroxisomal flavoenzyme that catalyzes the stereoselective oxidative deamination of D-amino acids to their α-keto acids, hydrogen peroxide and ammonia (Pilone 2000;Sacchi et al 2012). In peroxisomes, catalase subsequently ensures for the rapid elimination of the highly reactive hydrogen peroxide (Usuda et al 1986;Pollegioni et al 2007).…”

A conditional marker gene allowing both positive and negative selection in plants

“…Damino acid oxidase is a well-characterized enzyme, and both its crystal structure and its catalytic mechanism have been determined by high-resolution x-ray spectroscopy 5 . It is a flavoenzyme located in the peroxisome, and its recognised function in animals is detoxification of D-amino acids 2 . In addition, it gives yeasts the ability to use D-amino acids for growth 6 .…”