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

Abstract

Flavin is one of the most versatile redox cofactors in nature and is used by many enzymes to perform a multitude of chemical reactions. D-Amino acid oxidase (DAAO), a member of the flavoprotein oxidase family, is regarded as a key enzyme for the understanding of the mechanism underlying flavin catalysis. The very highresolution structures of yeast DAAO complexed with D-alanine, D-trifluoroalanine, and L-lactate (1.20, 1.47, and 1.72 Å) provide strong evidence for hydride transfer as the mechanism of dehydrogenation. This is inconsistent with the alternative carbanion mechanism originally favored for this type of enzymatic reaction. The step of hydride transfer can proceed without involvement of amino acid functional groups. These structures, together with results from site-directed mutagenesis, point to orbital orientation͞steering as the major factor in catalysis. A diatomic species, proposed to be a peroxide, is found at the active center and on the Re-side of the flavin. These results are of general relevance for the mechanisms of flavoproteins and lead to the proposal of a common dehydrogenation mechanism for oxidases and dehydrogenases. D-Amino acid oxidase (DAAO) was one of the first enzymes to be described and the second flavoprotein to be discovered in the mid 1930s (1, 2). It catalyzes the dehydrogenation of D-amino acids to their imino counterparts via the Michaelis complexes M1 and the reduced flavin-product complex M2 (Fig. 1). The reduced flavin is then (re)oxidized by dioxygen to yield FAD ox and H 2 O 2 , whereas the imino acid spontaneously hydrolyzes to the keto acid and NH 4 ϩ . Although DAAO is present in most organisms and mammalian tissues, its physiological role in vertebrates has been unclear (3). Most recently, however, a specific role of DAAO in the degradation of the neurotransmitter D-serine in brain has been proposed (4), consistent with a role in the regulation of neurotransmission.The dehydrogenation, catalyzed by the class of flavoprotein oxidases and dehydrogenases, as exemplified by DAAO, is a fundamental biochemical reaction. Despite this, its molecular mechanism is still a matter of dispute and has evoked several contrasting proposals over the years. In 1971, Walsh et al. (5) discovered that pig kidney DAAO (pkDAAO) catalyzes the elimination of halide from ␤-halogenated amino acids. This led to the seemingly reasonable conclusion, found in most biochemistry textbooks, that catalysis involves abstraction of the amino acid ␣H as H ϩ via the so-called ''carbanion mechanism,'' a process that requires an active site base. This concept was challenged in 1975, based on work with pkDAAO reconstituted with the artificial cofactor 5-deazaFAD by Hersh and SchumanJorns (6), who favored a hydride mechanism proceeding via transfer of the substrate ␣COH to the flavin N (5). These mechanisms, the carbanion and the hydride transfer, represent the two extremes under consideration for such a chemical oxidation reaction.Recently the three-dimensional structure of pkDAAO complexed with benzoate,...

Deflavination of flavo-oxidases by nucleophilic reagents

“…In particular, the 3D model of vanillyl-AO shows that a tyrosine (Tyr 187) is involved in a hydrogen bond with the £avin ring and that two other Tyr residues (Tyr 108 and 503) are part of the catalytic site of the enzyme [6]. Similar topology and participation of two tyrosine residues (Tyr 223 and 228) in the substrate-binding site of DAOX was also found in the very high-resolution structure of the enzyme [24]. The analysis of the FAD steady-state £uorescence, £uorescence and anisotropy decays of H. polymorpha and P. pastoris AO [14] also indicated interactions of their £avin moieties with aromatic side chains from the FAD-binding pocket which led to a strong quenching of the £avin £uorescence.…”

Effect of ligand binding on human D‐amino acid oxidase: Implications for the development of new drugs for schizophrenia treatment

“…Similarly, L-serine cannot be used since it is neither substrate or inhibitor of hDAAO. CF 3 -D-Ala is a pseudo-substrate of DAAO that was previously used to deep insight the mode of substrate binding in the yeast enzyme: 18 it possesses all the structural and steric requirements to be a substrate of DAAO but dehydrogenation is not feasible because of the inductive effect due to the ACF 3 side chain. In fact, the addition of CF 3 -D-Ala under anaerobic conditions does not convert the oxidized hDAAO into the reduced enzyme form, as instead observed using a classical substrate such as D-alanine.…”

Unfolding Intermediate in the Peroxisomal Flavoprotein d-Amino Acid Oxidase

“…The intrinsic fluorescence of tryptophan residues was used as probe of the unfolding of DAAO. Because there are eight tryptophans in yeast DAAO (11), the overall changes in fluorescence reflect global changes in protein structure, and only the average microenvironments of tryptophans can be assessed. As stated above, tryptophan emission at ϳ345 nm (following excitation at 280 or 298 nm) is significantly higher for ⌬ loop DAAO than for wild type.…”

“…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. The three-dimensional structure of DAAO has been resolved at very high resolution, allowing investigators to find the rationale of its high catalytic efficiency (11,12). In the "side to tail" model of monomer-monomer interaction (with a high buried surface area, 3049 Å 2 ) (12), a large contribution to the interaction between monomers is given by a long (21 amino acids) loop connecting ␤-strands F5 and F6, that is unique to yeast DAAO.…”