Roentgen-Struktur-Analyse von zwei loeslichen Proteinen mit Kofaktor: D-Aminosaeure Oxidase aus der Hefe Rhodotorula gracilis und dem Neun Haem Cytochrome c aus sulfatereduzierenden Bakterien Desulfovibrio desulfuricans Essex 6

The flavin ring-system constitutes 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 oxidase family, is regarded as a key enzyme for the understanding of the mechanism underlying flavin catalysis. The ultra-high resolution structure of yeast DAAO in complex with substrates allows the unambiguous identification of hydride transfer as the dehydrogenation mechanism. The hydride transfer mechanism proceeds without involvement of functional groups and points to orbital orientation as the major factor in catalysis. The results presented here are of general relevance for the mechanisms of flavoprotein oxidases and dehydrogenases and provide a unifying concept for flavin catalysis. The X-ray structure of nine heme cytochrome c from the sulfate reducing bacteria Desulfovibrio desulfuricans strain Essex 6 was solved by the multiple wavelength anomalous dispersion (MAD) phasing method. Nine heme cytochrome consists of two tetraheme cytochrome c3-like domains with an equivalent four heme arrangement. The two domains at the N- and C-terminus are connected by a ninth heme buried under the protein surface. It is held in position by loop extensions in the flanking domains. Detailed analysis of the three- dimensional structure allows to characterize their specialization in evolution for interacting in the most productive manner with their reaction partners. A positive charged patch at the surface of the C-terminal domain with a heme cofactor in its center is an optimal acceptor for electrons originating from hydrogenase. Two predominant loop extensions not found in tetraheme cytochrome c3 s in the N-terminal domain might enhance the contact to the membrane bound complex. This high molecular mass complex (hmc) is located in the cytoplasmic membrane and responsible for the transport of electrons from the periplasm to the

Prediction by a neural network of outer membrane beta-strand protein topology

An artificial neural network (NN) was trained to predict the topology of bacterial outer membrane (OM) β-strand proteins. Specifically, the NN predicts the z-coordinate of Cα 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 β-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 Cα 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

Three-dimensional structure of the nonaheme cytochrome c from desulfovibrio Desulfuricans essex in the Fe(III) state at 1.89 Å resolution

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 Å, and anisotropic temperature factors were addressed to the iron and sulfur atoms in the model. The structure revealed two cytochrome c3 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 c3, 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

Yeast D-amino acid oxidase : structural basis of its catalytic properties

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-CF3-alanine, or of L-lactate, in the anthrani-late 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 regu-late 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 tight-ness of FAD cofactor binding between the DAAOs from different sources

Yeast D-Amino acid oxidase : structural basis of its catalytic properties

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 1.9A) 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-CF3-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

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

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 high-resolution 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 orientation/steering 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