Biomedical subjects

Yoshiki Higuchi

Publications and source records attributed to Yoshiki Higuchi.

25 records · Page 2Linked to original sources

PubMed2003

Crystallization and preliminary neutron analysis of the dissimilatory sulfite reductase D (DsrD) protein from the sulfate-reducing bacterium Desulfovibrio vulgaris.

Dissimilatory sulfite reductase D (DsrD) from Desulfovibrio vulgaris has been crystallized for a neutron diffraction study. The initial crystals obtained were too small for the neutron experiment. In order to obtain a larger crystal (>1 mm3), a combination of two techniques was developed to determine the optimum crystallization conditions: a crystallization phase diagram was obtained, followed by crystal-quality assessment via X-ray diffraction. Using conditions determined in this manner, a large single crystal (1.7 mm3) of DsrD protein was subsequently grown in D(2)O solution by the macroseeding technique. A neutron diffraction experiment was carried out using the BIX-3 diffractometer at the Japan Atomic Energy Research Institute (JAERI), collecting data to 2.4 A resolution from an optimized crystal.

Crystallization↗

PubMed2003

X-ray crystallographic characterization and phasing of a fucose-specific lectin from Aleuria aurantia.

A fucose-specific lectin from Aleuria aurantia was crystallized in its native form and was also cocrystallized with HgCl(2). Crystallization was performed using the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant. Both the Hg-free native crystals and the Hg cocrystals belong to the hexagonal space group P6(5)22. The unit-cell parameters for the Hg-free form are a = 84.0, c = 250.1 A and for the Hg cocrystals are a = 83.9, c = 254.3 A. Both forms of the crystals diffract X-rays to 2.3 A resolution and are suitable for high-resolution crystal structure determination. Initial phasing was successfully performed by the MAD method using the Hg cocrystals and the electron density obtained was good enough for model building.

Ascomycota↗

PubMed2003

Single crystal EPR studies of the reduced active site of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F.

In the catalytic cycle of [NiFe] hydrogenase the paramagnetic Ni-C intermediate is of key importance, since it is believed to carry the substrate hydrogen, albeit in a yet unknown geometry. Upon illumination at low temperatures, Ni-C is converted to the so-called Ni-L state with markedly different spectroscopic parameters. It is suspected that Ni-L has lost the "substrate hydrogen". In this work, both paramagnetic states have been generated in single crystals obtained from the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. Evaluation of the orientation dependent spectra yielded the magnitudes of the g tensors and their orientations in the crystal axes system for both Ni-C and Ni-L. The g tensors could further be related to the atomic structure by comparison with the X-ray crystallographic structure of the reduced enzyme. Although the g tensor magnitudes of Ni-C and Ni-L are quite different, the orientations of the resulting g tensors are very similar but differ from those obtained earlier for Ni-A and Ni-B (Trofanchuk et al. J. Biol. Inorg. Chem. 2000, 5, 36-44). The g tensors were also calculated by density functional theory (DFT) methods using various structural models of the active site. The calculated g tensor of Ni-C is, concerning magnitudes and orientation, in good agreement with the experimental one for a formal Ni(III) oxidation state with a hydride (H(-)) bridge between the Ni and the Fe atom. Satisfying agreement is obtained for the Ni-L state when a formal Ni(I) oxidation state is assumed for this species with a proton (H(+)) removed from the bridge between the nickel and the iron atom.

Bacterial Proteins↗

PubMed2003

Role of the aromatic ring of Tyr43 in tetraheme cytochrome c(3) from Desulfovibrio vulgaris Miyazaki F.

Tyrosine 43 is positioned parallel to the fifth heme axial ligand, His34, of heme 1 in the tetraheme cytochrome c(3). The replacement of tyrosine with leucine increased the redox potential of heme 1 by 44 and 35 mV at the first and last reduction steps, respectively; its effects on the other hemes are small. In contrast, the Y43F mutation hardly changed the potentials. It shows that the aromatic ring at this position contributes to lowering the redox potential of heme 1 locally, although this cannot be the major contribution to the extremely low redox potentials of cytochrome c(3). Furthermore, temperature-dependent line-width broadening in partially reduced samples established that the aromatic ring at position 43 participates in the control of the kinetics of intramolecular electron transfer. The rate of reduction of Y43L cytochrome c(3) by 5-deazariboflavin semiquinone under partially reduced conditions was significantly different from that of the wild type in the last stage of the reduction, supporting the involvement of Tyr43 in regulation of reduction kinetics. The mutation of Y43L, however, did not induce a significant change in the crystal structure.

Amino Acid Substitution↗

PubMed2003

Crystal structure of dissimilatory sulfite reductase D (DsrD) protein--possible interaction with B- and Z-DNA by its winged-helix motif.

The crystal structure of DsrD from Desulfovibrio vulgaris Hildenborough has been determined at 1.2 A resolution. DsrD is in a dimeric form in the crystal, and five sulfate anions were located on the surface. The structure of DsrD comprises a winged-helix motif, which shows the highest structural homology to similar motifs found in Z-DNA binding proteins and some B-DNA binding proteins. The core structure of the molecule is constructed by intramolecular interactions of hydrophobic residues, which are well conserved in DNA binding proteins, suggesting that these proteins belong to the same superfamily on the basis of the structure. These results indicate a possible role of DsrD in transcription or translation of genes for enzymes catalyzing dissimilatory sulfite reduction.

Amino Acid Sequence↗

PubMed2002

Structural studies of the carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F: suggestion for the initial activation site for dihydrogen.

The carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F has been characterized by X-ray crystallography and absorption and resonance Raman spectroscopy. Nine crystal structures of the [NiFe]hydrogenase in the CO-bound and CO-liberated forms were determined at 1.2-1.4 A resolution. The exogenously added CO was assigned to be bound to the Ni atom at the Ni-Fe active site. The CO was not replaced with H(2) in the dark at 100 K, but was liberated by illumination with a strong white light. The Ni-C distances and Ni-C-O angles were about 1.77 A and 160 degrees, respectively, except for one case (1.72 A and 135 degrees ), in which an additional electron density peak between the CO and Sgamma(Cys546) was recognized. Distinct changes were observed in the electron density distribution of the Ni and Sgamma(Cys546) atoms between the CO-bound and CO-liberated structures for all the crystals tested. The novel structural features found near the Ni and Sgamma(Cys546) atoms suggest that these two atoms at the Ni-Fe active site play a role during the initial H(2)-binding process. Anaerobic addition of CO to dithionite-reduced [NiFe]hydrogenase led to a new absorption band at about 470 nm ( approximately 3000 M(-1)cm(-1)). Resonance Raman spectra (excitation at 476.5 nm) of the CO complex revealed CO-isotope-sensitive bands at 375/393 and 430 cm(-1) (368 and 413 cm(-1) for (13)C(18)O). The frequencies and relative intensities of the CO-related Raman bands indicated that the exogenous CO is bound to the Ni atom with a bent Ni-C-O structure in solution, in agreement with the refined structure determined by X-ray crystallography.

Binding Sites↗

PubMed2002

Extended lymphadenectomy and preoperative radiotherapy for lower rectal cancers.

BACKGROUND: Extended lymphadenectomy including lateral node dissection (EXT-L) contributes to a low incidence of local recurrence of lower rectal cancer. However, EXT-L is frequently associated with impairment of sexual and urinary function. We therefore compared the effectiveness of preoperative radiotherapy with that of EXT-L. METHODS: One hundred fifteen patients were studied. Seventy-eight patients underwent preoperative radiotherapy with a total dose of 50 Gy (Rad[+] group), and 37 did not (Rad[-] group). Seventy-five patients received EXT-L (EXT-L[+] group), and 40 did not (EXT-L[-] group). Patients were further divided into 4 subgroups (Rad[+]&EXT-L[-], Rad(+)&EXT-L[+], Rad[-]&EXT-L(+), and Rad[-]&EXT-L[-]), and clinicopathologic features were examined. In the Rad(+) group, the relation between the p53 gene and survival was also examined. RESULTS: There was a significant difference in disease-free survival between the Rad(+) and Rad(-) groups (5-year disease-free survival rate, 74.6% vs 45.9%; P =.006). However, there was no significant difference between the Rad(+)&EXT-L[-] and Rad[-]&EXT-L(+) groups. The p53 gene status did not affect survival in the Rad(+) group. CONCLUSIONS: This study suggests that in terms of curative effect, preoperative radiotherapy can be one alternative therapy in place of EXT-L for patients with lower rectal cancer.

Adenocarcinoma↗