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,...

The 3D structure of the flavoprotein D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis (RgDAAO) in complex with the competitive inhibitor anthranilate was solved (resolution 1.9 Å ) and structural features relevant for the overall conformation and for catalytic activity are described. The FAD is bound in an elongated conformation in the core of the enzyme. Two anthranilate molecules are found within the active site cavity; one is located in a funnel forming the entrance, and the second is in contact with the flavin. The anchoring of the ligand carboxylate with Arg285 and Tyr223 is found for all complexes studied. However, while the active site group Tyr238-OH interacts with the carboxylate in the case of the substrate D-alanine, of D-CF 3 -alanine, or of L-lactate, in the anthranilate complex the phenol group rotates around the C2 -C3 bond thus opening the entrance of the active site, and interacts there with the second bound anthranilate. This movement serves in channeling substrate to the bottom of the active site, the locus of chemical catalysis. The absence in RgDAAO of the "lid" covering the active site, as found in mammalian DAAO, is interpreted as being at the origin of the differences in kinetic mechanism between the two enzymes. This lid has been proposed to regulate product dissociation in the latter, while the side-chain of Tyr238 might exert a similar role in RgDAAO. The more open active site architecture of RgDAAO is the origin of its much broader substrate specificity. The RgDAAO enzyme forms a homodimer with C2 symmetry that is different from that reported for mammalian D-amino acid oxidase. This different mode of aggregation probably causes the differences in stability and tightness of FAD cofactor binding between the DAAOs from different sources.

An artificial neural network (NN) was trained to predict the topology of bacterial outer membrane (OM) beta-strand proteins. Specifically, the NN predicts the z-coordinate of Calpha atoms in a coordinate frame with the outer membrane in the xy-plane, such that low z-values indicate periplasmic turns, medium z-values indicate transmembrane beta-strands, and high z-values indicate extracellular loops. To obtain a training set, seven OM proteins (porins) with structures known to high resolution were aligned with their pores along the z-axis. The relationship between Calpha z-values and topology was thereby established. To predict the topology of other OM proteins, all seven porins were used for the training set. Z-values (topologies) were predicted for two porins with hitherto unknown structure and for OM proteins not belonging to the porin family, all with insignificant sequence homology to the training set. The results of topology prediction compare favorably with experimental topology data.

A nine heme group containing cytochrome c isolated from the soluble and membrane fractions of Desulfovibrio desulfuricans Essex, termed nonaheme cytochrome c, was crystallized, and the structure was solved using the multiple wavelength anomalous dispersion (MAD) phasing method. Refinement was carried out to a resolution of 1.89 A, and anisotropic temperature factors were addressed to the iron and sulfur atoms in the model. The structure revealed two cytochrome c(3) like domains with the typical arrangement of four heme centers. Both domains flanked an extra heme buried under the protein surface. This heme is held in position by loop extensions in each of the two domains. Although both the N- and C-terminal tetraheme domains exhibit a fold and heme arrangement very similar to that of cytochrome c(3), they differ considerably in their loop extensions and electrostatic surface. Analysis of the structure provides evidence for a different function of both domains, namely, anchoring the protein in a transmembranous complex with the N-terminal domain and formation of an electron-transfer complex with hydrogenase by the C-terminal domain.