Three-Dimensional Structure of Porcine Kidney D-Amino Acid Oxidase at 3.0 A Resolution

Mizutani, H.; Miyahara, Ikuko; Hirotsu, Ken; Nishina, Yasuzo; Shiga, Kiyoshi; Setoyama, Chiaki; Miura, Retsu

doi:10.1093/oxfordjournals.jbchem.a021376

Cited by 120 publications

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“…A comparison of flavin enzymes dehydrogenating ␣-OH and ␣-amino acids is thus instructive and leads to the formulation of a common mechanism. These two subclasses of enzymes have long been assumed to be related, and the finding of major differences in the nature and presence of functional groups at the active centers has been puzzling (7,9). In flavocytochrome b 2 and glycollate oxidase (26,27), a lysine-NH 3 ϩ close to the flavin pyrimidine N(1) stabilizes the charge developing on the flavin on reduction.…”

Section: General Mechanisms Of Substrate Dehydrogenation and Dioxygenmentioning

confidence: 99%

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

Umhau

Pollegioni

Molla

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

180

221

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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,...

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Section: General Mechanisms Of Substrate Dehydrogenation and Dioxygenmentioning

confidence: 99%

“…Recently the three-dimensional structure of pkDAAO complexed with benzoate, iminotryptophane, and o-aminobenzoate at 2.5-to 3.0-Å resolution was reported by two independent groups (7)(8)(9)(10). The mechanistic interpretations of the structural data were quite contrasting.…”

mentioning

confidence: 99%

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

Umhau

Pollegioni

Molla

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

180

221

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“…The experimentally determined structural coordinates of DAOs (PDB entry code: 1evi, 1an9, and 1aa8) 18,21,22 were used as references for model building. The backbone atom positions of these template structures were averaged.…”

Section: Methodsmentioning

confidence: 99%

“…Very recently, these molecular details have been solved by X-ray crystallography 18 . We have cloned a gene encoding DAO from the hepatopancreas of carp (Cyprinus carpio) for the first time in a non-mammalian species (GenBank accession no.…”

Section: Introductionmentioning

confidence: 99%

Untitled

Sarower¹,

Okada²,

Abe³

2009

ScienceAsia

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ABSTRACT:The three-dimensional structure of the flavoprotein D-amino acid oxidase (DAO, EC 1.4.3.3) from carp hepatopancreas (chDAO) and its active site cavity was modelled using ProModII. The structural features relevant for the overall conformation and for the catalytic activity are described. Secondary structure topology consists of 11 α-helices and 17 β-strands, which differs slightly from pig kidney, and Rhodotorula gracilis DAOs. chDAO showed a theoretical 'head-to-head' mode of dimerization. The presence of a short 'lid' in chDAO covering the active site, commonly found in mammalian DAO but absent in R. gracilis DAO, is interpreted as the origin of the differences in kinetic mechanism among these enzymes. This lid has been proposed to control the access of the substrate to the active site and to regulate dissociation of products. The conformational change in the large size active site loop determines the overall rate of turnover of DAOs. The shorter active site loop found in chDAO might be responsible for the higher turnover rate in chDAO compared to that of pkDAO.

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“…Moreover, D-amino acid oxidase, which belongs to a different enzyme family but carries out a rather similar chemical reaction, was thought to also use a carbanion mechanism (Bright & Porter, 1975;Ghisla, 1982;Denu & Fitzpatrick, 1994); nevertheless, its recently published crystal structure appears to rule out such a possibility and may be compatible with a hydride transfer reaction (Mattevi et al. 1996;Mizutani et al, 1996). As a consequence, it was recently suggested that the accepted mechanism of the a-hydroxy acidoxidizing enzymes might also need revision (Pollegioni et al, 1997;Yorita et al, 1997).…”

Section: H373 K349mentioning

confidence: 99%

About the pK_a of the active‐site histidine in flavocytochrome b₂ (yeast L‐lactate dehydrogenase)

Rao

Lederer

1998

Protein Science

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Flavocytochrome b2 or L-lactate dehydrogenase from yeasts catalyzes the oxidation of L-lactate at the expense of monoelectronic acceptors such as cytochrome c, its physiological partner. When incubated in the presence of both L-lactate and a keto acid, the enzyme catalyzes a transhydrogenation reaction wherein only the flavin is involved. During this reaction, the substrate a-hydrogen is transferred not only to the solvent but also in part to the keto acid, which acts as reverse substrate. Thus, when bound to the reduced enzyme, this hydrogen is sticky. In the context of a carbanion mechanism, it resides on NE of His373, the active site base. We have shown before that a correlation between the amount of intermolecular hydrogen transfer from [2-'H] lactate and the keto acid reverse substrate concentration enables the determination of the first-order rate constant, k:, for exchange of the substrate-derived protein-bound hydrogen with bulk solvent (Urban P, Lederer F, 1985, J BioZ Chem 260: 1 1 1 15-1 1 122). In this work, we show that the exchange with the solvent appears to be independent of the phosphate buffer concentration in the range from 40 to 500 mM. It is thus probable that exchange occurs directly with water molecules. The second-order rate constant for exchange is then 0.16 (kO.03) M" s". Using the Eigen equation, this figure yields a pK, of 9.1 f 0.1 for His373 in the reduced enzyme, compared to a probable value of 6.0 or less in the oxidized enzyme (Suzuki H, Ogura YC, 1970, JBiochem 67:291-295). The mechanistic significance of these results is discussed.

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Three-Dimensional Structure of Porcine Kidney D-Amino Acid Oxidase at 3.0 A Resolution

Cited by 120 publications

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

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

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About the pK_a of the active‐site histidine in flavocytochrome b₂ (yeast L‐lactate dehydrogenase)

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Three-Dimensional Structure of Porcine Kidney D-Amino Acid Oxidase at 3.0 A Resolution

Cited by 120 publications

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

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

Untitled

About the pKa of the active‐site histidine in flavocytochrome b2 (yeast L‐lactate dehydrogenase)

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About the pK_a of the active‐site histidine in flavocytochrome b₂ (yeast L‐lactate dehydrogenase)