D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of Damino acids to the corresponding a-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that ofp-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate a-hydrogen to the flavin during the enzyme reductive half-reaction. The active site of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups of these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalyze similar reactions (oxidation of a-amino acids or a-hydroxy acids), although with opposite stereochemistry.Since the description of D-amino acid oxidase (EC 1.4.3.3; DAAO) activity in mammalian tissues by Krebs in 1935 (1), DAAO has been the subject of a number of biochemical, spectroscopic, and kinetic investigations, becoming the prototype for the oxidase class of the flavin-containing enzymes [for a recent review, see ref. 2]. Its primary structure has been determined and its gene has been cloned (3, 4). Its kinetic and mechanistic properties have been studied in detail by a variety of techniques, while information on the topology of the active site and on its three-dimensional structure have only been derived from chemical modification studies and site-directed mutagenesis of selected residues. Based on these approaches, a catalytic mechanism for DAAO has been proposed, although definitive evidence against alternative mechanisms has not been found (refs. 2 and 5 and references therein).The enzyme catalyzes the oxidation of D-a-amino acids into the corresponding a-ketoacids. The reaction formally proceeds according to the following scheme:E-FADH2 + 02-*E-FAD + H202[2]The reductive half reaction (Eq. 1), in which the noncovalently bound FAD becomes reduced, is followed by the oxidative step in which FAD is reoxidized by molecular oxygen, with the release of hydrogen peroxide (Eq. 2). The imino acid product spontaneously hydrolyzes to the ketoacid in a nonenzymatic process (Eq. 3). DAAO displays a broad substrate specificity, with a preference for D-amin...

The shape of the active-site cavity controls substrate specificity by providing a 'size exclusion mechanism'. Inside the cavity, the substrate aromatic ring is positioned at an angle of 18 degrees to the flavin ring. This arrangement is ideally suited for a hydride transfer reaction, which is further facilitated by substrate deprotonation. Burying the substrate beneath the protein surface is a recurrent strategy, common to many flavoenzymes that effect substrate oxidation or reduction via hydride transfer.

By mutating the target residue of covalent flavinylation in vanillyl-alcohol oxidase, the functional role of the histidyl-FAD bond was studied. Three His 422 mutants (H422A, H422T, and H422C) were purified, which all contained tightly but noncovalently bound FAD. Steady state kinetics revealed that the mutants have retained enzyme activity, although the turnover rates have decreased by 1 order of magnitude. Stopped-flow analysis showed that the H422A mutant is still able to form a stable binary complex of reduced enzyme and a quinone methide product intermediate, a crucial step during vanillyl-alcohol oxidase-mediated catalysis. The only significant change in the catalytic cycle of the H422A mutant is a marked decrease in reduction rate. Redox potentials of both wild type and H422A vanillylalcohol oxidase have been determined. During reduction of H422A, a large portion of the neutral flavin semiquinone is observed. Using suitable reference dyes, the redox potentials for the two one-electron couples have been determined: ؊17 and ؊113 mV. Reduction of wild type enzyme did not result in any formation of flavin semiquinone and revealed a remarkably high redox potential of ؉55 mV. The marked decrease in redox potential caused by the missing covalent histidyl-FAD bond is reflected in the reduced rate of substrate-mediated flavin reduction limiting the turnover rate.Elucidation of the crystal structure of the H422A mutant established that deletion of the histidyl-FAD bond did not result in any significant structural changes. These results clearly indicate that covalent interaction of the isoalloxazine ring with the protein moiety can markedly increase the redox potential of the flavin cofactor, thereby facilitating redox catalysis. Thus, formation of a histidyl-FAD bond in specific flavoenzymes might have evolved as a way to contribute to the enhancement of their oxidative power.Until now, several hundred flavin-containing enzymes have been described. Most of these enzymes contain a dissociable FAD or FMN cofactor. However, it has been shown that in several cases the flavin is covalently linked to an amino acid of the polypeptide chain. In fact, in humans 10% of the cellular FAD is covalently bound to enzymes like e.g. succinate dehydrogenase and monoamine oxidase (1). Within the group of covalent flavoproteins, five different types of covalent flavinylation have been identified. Except for a few examples of cysteinyl-or tyrosyl-linked flavins, tethering to a histidine is by far the most favored binding mode, since it has been observed in about 20 isolated flavoenzymes (for a recent review, see Ref. 2).Although the first covalent flavoprotein, succinate dehydrogenase, was already identified in 1955 (3), the rationale for covalent flavinylation is still unresolved. Only recently, a clear influence of the covalent bond on the reactivity of the cofactor has been observed in trimethylamine dehydrogenase. Unlike the wild type enzyme, mutants of trimethylamine dehydrogenase containing dissociable FMN (4, 5) are inactivated b...

The catalytic domain of dihydrolipoyl transacetylase (E2pCD) forms the core of the pyruvate dehydrogenase multienzyme complex and catalyzes the acetyltransferase reaction using acetylCoA as acetyl donor and dihydrolipoamide (Lip(SH)2) as acceptor. The crystal structures of six complexes and derivatives of Azotobacter vinelandii E2pCD were solved. The binary complexes of the enzyme with CoA and Lip(SH)2 were determined at 2.6- and 3.0-A resolutions, respectively. The two substrates are found in an extended conformation at the two opposite entrances of the 30 A long channel which runs at the interface between two 3-fold-related subunits and forms the catalytic center. The reactive thiol groups of both substrates are within hydrogen-bond distance from the side chain of His 610. This fact supports the indication, derived from the similarity with chloramphenicol acetyl transferase, that the histidine side chain acts as general-base catalyst in the deprotonation of the reactive thiol of CoA. The conformation of Asn 614 appears to be dependent on the protonation state of the active site histidine, whose function as base catalyst is modulated in this way. Studies on E2pCD soaked in a high concentration of dithionite lead to the structure of the binary complex between E2pCD and hydrogen sulfite solved at 2.3-A resolution. It appears that the anion is bound in the middle of the catalytic center and is therefore capable of hosting and stabilizing a negative charge, which is of special interest since the reaction catalyzed by E2pCD is thought to proceed via a negatively charged tetrahedral intermediate. The structure of the binary complex between E2pCD and hydrogen sulfite suggests that transition-state stabilization can be provided by a direct hydrogen bond between the side chain of Ser 558 and the oxy anion of the putative intermediate. In the binary complex with CoA, the hydroxyl group of Ser 558 is hydrogen bonded to the nitrogen atom of one of the two peptide-like units of the substrate. Thus, CoA itself is involved in keeping the Ser hydroxyl group in the proper position for transition-state stabilization. Quite unexpectedly, the structure at 2.6-A resolution of a ternary complex in which CoA and Lip(SH)2 are simultaneously bound to E2pCD reveals that CoA has an alternative, nonproductive binding mode. In this abortive ternary complex, CoA adopts a helical conformation with two intramolecular hydrogen bonds and the reactive sulfur of the pantetheine arm positioned 12 A away from the active site residues involved in the transferase reaction.(ABSTRACT TRUNCATED AT 400 WORDS)

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