Noritake Yasuoka

The Structure of the Mammalian 20S Proteasome at 2.75 Å Resolution

The 20S proteasome is the catalytic portion of the 26S proteasome. Constitutively expressed mammalian 20S proteasomes have three active subunits, beta 1, beta 2, and beta 5, which are replaced in the immunoproteasome by interferon-gamma-inducible subunits beta 1i, beta 2i, and beta 5i, respectively. Here we determined the crystal structure of the bovine 20S proteasome at 2.75 A resolution. The structures of alpha 2, beta 1, beta 5, beta 6, and beta 7 subunits of the bovine enzyme were different from the yeast enzyme but enabled the bovine proteasome to accommodate either the constitutive or the inducible subunits. A novel N-terminal nucleophile hydrolase activity was proposed for the beta 7 subunit. We also determined the site of the nuclear localization signals in the molecule. A model of the immunoproteasome was predicted from this constitutive structure.

Unusual ligand structure in Ni-Fe active center and an additional Mg site in hydrogenase revealed by high resolution X-ray structure analysis

BACKGROUND The hydrogenase of Desulfovibrio sp. catalyzes the reversible oxidoreduction of molecular hydrogen, in conjunction with a specific electron acceptor, cytochrome c3. The Ni-Fe active center of Desulfovibrio hydrogenase has an unusual ligand structure with non-protein ligands. An atomic model at high resolution is required to make concrete assignment of the ligands which coordinate the Ni-Fe center. These in turn will provide insight into the mechanism of electron transfer, during the reaction catalysed by hydrogenase. RESULTS The X-ray structure of the hydrogenase from Desulfovibrio vulgaris Miyazaki has been solved at 1.8 A resolution and refined to a crystallographic R factor of 0.229. The overall folding pattern and the spatial arrangement of the metal centers are very similar to those found in Desulfovibrio gigas hydrogenase. This high resolution crystal structure enabled us to assign the non-protein ligands to the Fe atom in the Ni-Fe site and revealed the presence of a Mg center, located approximately 13 A from the Ni-Fe active center. CONCLUSIONS From the nature of the electron-density map, stereochemical geometry and atomic parameters of the refined structure, the most probable candidates for the four ligands, coordinating the Ni-Fe center, have been proposed to be diatomic S=O, C triple bond O and C triple bond N molecules and one sulfur atom. The assignment was supported by pyrolysis mass spectrometry measurements. These ligands may have a role as an electron sink during the electron transfer reaction between the hydrogenase and its biological counterparts, and they could stabilize the redox state of Fe(II), which may not change during the catalytic cycle and is independent of the redox transition of the Ni. The hydrogen-bonding system between the Ni-Fe and the Mg centers suggests the possible.

Removal of the bridging ligand atom at the Ni–Fe active site of [NiFe] hydrogenase upon reduction with H2, as revealed by X-ray structure analysis at 1.4 Å resolution

BACKGROUND The active site of [NiFe] hydrogenase, a heterodimeric protein, is suggested to be a binuclear Ni-Fe complex having three diatomic ligands to the Fe atom and three bridging ligands between the Fe and Ni atoms in the oxidized form of the enzyme. Two of the bridging ligands are thiolate sidechains of cysteinyl residues of the large subunit, but the third bridging ligand was assigned as a non-protein monatomic sulfur species in Desulfovibrio vulgaris Miyazaki F hydrogenase. RESULTS The X-ray crystal structure of the reduced form of D. vulgaris Miyazaki F [NiFe] hydrogenase has been solved at 1.4 A resolution and refined to a crystallographic R factor of 21.8%. The overall structure is very similar to that of the oxidized form, with the exception that the third monatomic bridge observed at the Ni-Fe site in the oxidized enzyme is absent, leaving this site unoccupied in the reduced form. CONCLUSIONS The unusual ligand structure found in the oxidized form of D. vulgaris Miyazaki F [NiFe] hydrogenase was confirmed in the reduced form of the enzyme, with the exception that the electron density assigned to the monatomic sulfur bridge had almost disappeared. On the basis of this finding, as well as the observation that H2S is liberated from the oxidized enzyme under an atmosphere of H2 in the presence of its electron carrier, it was postulated that the monatomic sulfur bridge must be removed for the enzyme to be activated. A possible mechanism for the catalytic action of the hydrogenase is proposed.

Refined structure of cytochrome c3 at 1.8 Å resolution

The structure of cytochrome c3 from the sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki has been successfully refined at 1.8 A resolution. The crystallographic R factor is 0.176 for 9907 significant reflections. The isotropic temperature factors of individual atoms were refined and a total of 47 water molecules located on the difference map were incorporated in the refinement. The four heme groups are closely packed, with adjacent pairs of heme planes being nearly perpendicular to each other. The fifth and the sixth ligands of the heme iron atoms are histidine residues with N epsilon 2-Fe distances ranging from 1.88 A to 2.12 A. The histidine co-ordination to the heme iron is different for each heme group. The heme groups are all highly exposed to solvent, although the actual regions exposed differ among the hemes. The four heme groups are located in different environments, and the heme planes are deformed from planarity. The differences in the heme structures and their environments indicate that the four heme groups are non-equivalent. The chemical as well as the physical properties of cytochrome c3 should be interpreted in terms of the structural non-equivalence of the heme groups. The characteristic secondary structural non-equivalence of the heme groups. The characteristic secondary structures of the polypeptide chain of this molecule are three short alpha-helices, two short beta-strands and ten reverse turns.

Crystal structures of ethanolamine ammonia-lyase complexed with coenzyme B12 analogs and substrates

N-terminal truncation of the Escherichia coli ethanolamine ammonia-lyase β-subunit does not affect the catalytic properties of the enzyme (Akita, K., Hieda, N., Baba, N., Kawaguchi, S., Sakamoto, H., Nakanishi, Y., Yamanishi, M., Mori, K., and Toraya, T. (2010) J. Biochem. 147, 83–93). The binary complex of the truncated enzyme with cyanocobalamin and the ternary complex with cyanocobalamin or adeninylpentylcobalamin and substrates were crystallized, and their x-ray structures were analyzed. The enzyme exists as a trimer of the (αβ)2 dimer. The active site is in the (β/α)8 barrel of the α-subunit; the β-subunit covers the lower part of the cobalamin that is bound in the interface of the α- and β-subunits. The structure complexed with adeninylpentylcobalamin revealed the presence of an adenine ring-binding pocket in the enzyme that accommodates the adenine moiety through a hydrogen bond network. The substrate is bound by six hydrogen bonds with active-site residues. Argα160 contributes to substrate binding most likely by hydrogen bonding with the O1 atom. The modeling study implies that marked angular strains and tensile forces induced by tight enzyme-coenzyme interactions are responsible for breaking the coenzyme Co–C bond. The coenzyme adenosyl radical in the productive conformation was modeled by superimposing its adenine ring on the adenine ring-binding site followed by ribosyl rotation around the N-glycosidic bond. A major structural change upon substrate binding was not observed with this particular enzyme. Gluα287, one of the substrate-binding residues, has a direct contact with the ribose group of the modeled adenosylcobalamin, which may contribute to the substrate-induced additional labilization of the Co–C bond.

The crystal and molecular structure of aceytlacetonatoacetylacetonyltriphenylphosphinepalladium(II) benzene solvate

Abstract The molecular structure of acetylacetonatoacetylacetonyltriphenylphosphinepalladium(II) [Pd(acad) 2 (PPh 3 )], has been determined by means of X-ray diffraction. The benzene solvate [Pd(acac) 2 (PPh 3 ·0.5C 6 H 6 ] forms yellow, monoclinic crystals; a = 36.444(8), b = 11.836(2), c = 16.170(4) », and β = 124.70(4) o , space group C 2/ c with Z = 8. The structure, solved by the conventional heavy-atom method, has been refined anisotropically by least-squares procedure to R = 0.078, for the 2806 independent non-zero reflections. The palladium atom takes a square-planar coordination. One of the acetylacetone ligands is bonded to the palladium via its oxygen atoms [Pd-O 2.048(10) and 2.062(10) »] and the other by its γ-carbon atom [Pd-C 2.114(13) »].

The crystal and molecular structure of a 3:2 mixture of laminarabiose and O-α-d-glucopyranosyl-(1→3)-β-d-glucopyranose

Abstract The crystal and molecular structure of a 3:2 mixture of laminarabiose and 3-O-α- d -glucopyranosyl-β- d -glucopyranose has been determined by X-ray diffraction. The crystal belongs to the monoclinic system, space group P2, a 14.778(1), b 4.794(1), c 10.516(1) A and β 98.10(1)c, Dm 1.54 g. cm-3, Z 2. The structure was solved by the direct method and refined by the block-diagonal, least-squares procedure to R 0.057 for 1034 observed reflections. Difference synthesis showed all hydrogen atoms and indicated a partial (∼39%), random substitution of the β anomer molecules by the α anomer molecules, which are accompanied by water molecules on the crystallographic two-fold axis (∼19%). The molecule shows a conformation, different from the fully-extended one, which is stabilized by an intramolecular hydrogen-bond between O-1-H and O-5 [2.786(7) A]. The ring-to-ring conformation can be described as (Φ, Ψ)=(27.9·–37.5·), according to the definition of Sathyanarayana and Rao, and it is located in the comparatively low-energy region of the energy-contour diagram of laminarabiose. Four intermolecular hydrogen-bonds hold molecules together to form infinite sheets, which are approximately parallel to the ab-plane and linked by additional hydrogen-bonds in the c-direction.

The Crystal and Molecular Structure of trans -Bis(trimethylphosphine)propynyl-1-(4′-dicyanomethylene-cyclohexa-2′-5′-dien-1-ylidenl-3,3-dicyano-2-methyl-prop-2-en-1-ylplatinum, a Reaction Product of trans -Bis(trimethylphosphine)bis(propynyl)platinum and 7,7,8,8-Tetracyanoquinodimethane

The molecular structure of the title complex has been determined from three-dimensional X-ray diffraction data. The crystal belongs to the monoclinic system, space group P21/c, a=14.934(2), b=8.997(1), c=20.221(3)A, β=97.44(2)°, Z=4. The molecular structure showed that the tetracyanoquinodimethane (TCNQ) molecule has reacted with one of the propynyl groups of trans-{(PMe3)2Pt(C≡CMe)2} : part of the TCNQ is bonded to the α-carbon atom and the other part to the β-carbon of the propynyl ligand. The platinum atom has a squareplanar geometry; Pt–P=2.302(5) and 2.290(5), Pt–C(C≡CMe3)=2.009(15) and Pt–C(reacted)=2.057(15) A.

Isolation and crystallization of high molecular weight cytochrome from Desulfovibrio valgaris Hildenborough

Abstract High molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough has been isolated, purified and crystallized. The molecular weight was estimated to be 75 000 as the mean value from the results of gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains 16 c-type heme groups per molecule. The purified high molecular weight cytochrome c shows typical c-type cytochrome absorption spectra, with an α-peak at 553.2 nm (ϵ553.2mM = 428) in the ferro form. The amino acid composition shows that the protein contains a sufficient number of cysteine and histidine residues to account for the high content of heme groups. The amino acid composition of D. vulgaris Hildenborough high molecular weight cytochrome c has no similarities to the D. desulfuricans hexahemoprotein nitrite reductase. The high content of the heme groups might suggest that the cytochrome has an alternate intrinsic biological function. Crystals of the high molecular weight cytochrome c have been grown from solutions of poly(ethylene glycol) 1000 or 2-methyl-2,4-pentanediol. The crystals are in space group P62 or P64 with unit cell dimensions a = b = 227.8, c = 105.7 A and γ = 120°. A large unit cell volume of 4.75 · 106 A3 suggests that there are four or five protein molecules per asymmetric unit. They diffract to better than 4.0 A resolution and appear to be resistant to radiation damage.

Single crystal EPR study of the Ni center of NiFe hydrogenase

Abstract EPR spectra of single crystals of NiFe hydrogenase from Desulfovibrio vulgaris Miyazaki F were evaluated and yielded the g-tensors of the Ni center for two different states of enzyme. The g-values associated with these states are identical to those measured in frozen solutions for the ready (NiB) and the unready (NiA) form of the Ni center. Directions of the g-tensor axes were determined relative to the crystal symmetry axes. The obtained changes of g-values and tensor axes orientations between NiA and NiB can be explained by a structural difference involving modification of a cysteine sulfur ligand.