Role of Arginine 285 in the Active Site of Rhodotorula gracilis d-Amino Acid Oxidase

Abstract

(1, 2). The precise mechanism of substrate dehydrogenation of this well studied enzyme (3) has not yet been solved, even if recently two groups have reported the crystal structure of the enzyme purified from pig kidney (pkDAAO) at a resolution of 2.6 and 3.0 Å, respectively (4, 5). Over the years, three main different mechanisms have been proposed for the reaction catalyzed by this flavoenzyme (see Mattevi et al. (6) for a recent review). (i) The hypothesis that the reductive half-reaction of DAAO involves the initial formation of a carbanion by abstraction of the ␣-H of the substrate as a proton comes from the elimination of halide from ␤-chloro-D-alanine (7). (ii) The observation of a transfer of ␣-hydrogen of the substrate to the C(5) position of the enzyme reconstituted with 5-deaza-FAD provides evidence in favor of a direct hydride-transfer mechanism (8). (iii) Finally a concerted mechanism (consistent with the experimental evidence for a carbanion mechanism) in which ␣-H ϩ abstraction is coupled with the transfer of a hydride from the amino group of the substrate has been put forward (9).As a model for DAAO to have a better understanding of this crucial issue, we have used the enzyme from the red yeast Rhodotorula gracilis (RgDAAO). Actually, RgDAAO possesses peculiar properties, as the high catalytic efficiency and the tight binding with the coenzyme FAD, which distinguish it from the mammalian enzyme (10 -12). These properties are most probably related to its physiological role (yeast can metabolize D-amino acids and use them as the sole nitrogen and carbon source) and to an evolutionary drive. From comparison of the primary sequences of the known DAAOs, it is evident that only three residues, among those identified in or near the active site (13), are conserved (namely two tyrosines and one arginine). The presence of an arginine residue located at the active site and directly involved in DAAO catalysis was in fact previously proposed by various chemical modification studies (for a review, see Ref. 3 and references therein). The possibility that the arginine residue could be involved in substrate binding by electrostatic interaction with the substrate was inferred by Nishino et al. (14) from 2,3-butanedione modification of the mammalian enzyme, followed by reaction with dansylchloride. On the other hand, the reactivity with sulfite and the spectral properties of the native and cyclohexandione-modified pk-DAAO reconstituted with 8-mercapto-FAD suggested that the active site arginine could act as the positively charged group near the flavin N(1)-C(2)ϭO locus and responsible for stabilization of anionic flavin forms (15). This basic residue has been identified in the primary sequence of RgDAAO, by irreversible inhibition using phenylglyoxal (16), although the question regarding its role was not solved. More recently, the resolution of

Glutamate receptor activation triggers a calcium-dependent and SNARE protein-dependent release of the gliotransmitter D-serine

“…The mutant enzyme shows a residual activity of Ͻ0.1% of that determined for the wild-type DAAO (general properties of Arg-285 3 Ala RgDAAO are described in ref. 15). The presence of the HRP and luminol does not affect the activity of the RgDAAO (see Fig.…”

The x-ray structure of d -amino acid oxidase at very high resolution identifies the chemical mechanism of flavin-dependent substrate dehydrogenation

“…In RgDAAO, such a group is absent; however, we propose that the same stabilization is exerted by the product ␣ANH 2 ϩ . Arg-285 appears to be involved in the stabilization of negatively charged flavin in the absence of ligands (28). It should be noted that with DAAOs, an (additional) stabilization of negatively charged flavin is observed also in the presence of carboxylate ligands that neutralize Arg-285 (3).…”

Analysis of the Role of the Active Site Residue Arg98 in the Flavoprotein Tryptophan 2-Monooxygenase, a Member of the l-Amino Oxidase Family

“…Biophys. 402,[24][25][26][27][28][29][30]. On the basis of the sequence comparisons of the different LAAO family members, Arg98 of tryptophan 2-monooxygenase can be identified as an active site residue which interacts with the carboxylate of the amino acid substrate.…”