Biomedical subjects

Nobuhumi Nakamura

Publications and source records attributed to Nobuhumi Nakamura.

6 recordsLinked to original sources

PubMed2005

DNA strands robed with ionic liquid moiety.

An ionic liquid domain was successfully prepared outside double-stranded DNA by fixing 1-alkyl-3-methyl-imidazolium (C(n)MI) cations on the phosphate groups of DNA. First, four species of ionic liquid were made using phosphoric acid di-n-butyl ester and C(n)MI (n=2,4,8, and 12) as a low molecular weight model. They were obtained as liquid salts, and their ionic conductivity ranged up to 10(-5)Scm(-1) at 50 degrees C. Based on this model study, counter cations of the phosphate groups of DNA were exchanged for four kinds of imidazolium cations. The resulting ionic liquid-robed DNA (IL-robed DNA) was soluble in ordinary organic solvents such as methanol or ethanol. Ionic conductivity was low, because the ion density was insufficient to form a continuous ionic liquid domain around the DNA strands. When 11mol% 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIBF(4)), which is a typical ionic liquid, was mixed with the IL-robed DNA, an ionic conductivity of 5.4 x 10(-5) S cm(-1) at 30 degrees C was observed because a continuous ionic liquid domain was successfully formed.

Coated Materials, Biocompatible↗

PubMed2005

Electron transfer chain reaction of the extracellular flavocytochrome cellobiose dehydrogenase from the basidiomycete Phanerochaete chrysosporium.

Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome containing flavin and b-type heme, and plays a key role in cellulose degradation by filamentous fungi. To investigate intermolecular electron transfer from CDH to cytochrome c, Phe166, which is located in the cytochrome domain and approaches one of propionates of heme, was mutated to Tyr, and the thermodynamic and kinetic properties of the mutant (F166Y) were compared with those of the wild-type (WT) enzyme. The mid-point potential of heme in F166Y was measured by cyclic voltammetry, and was estimated to be 25 mV lower than that of WT at pH 4.0. Although presteady-state reduction of flavin was not affected by the mutation, the rate of subsequent electron transfer from flavin to heme was halved in F166Y. When WT or F166Y was reduced with cellobiose and then mixed with cytochrome c, heme re-oxidation and cytochrome c reduction occurred synchronously, suggesting that the initial electron is transferred from reduced heme to cytochrome c. Moreover, in both enzymes the observed rate of the initial phase of cytochrome c reduction was concentration dependent, whereas the second phase of cytochrome c reduction was dependent on the rate of electron transfer from flavin to heme, but not on the cytochrome c concentration. In addition, the electron transfer rate from flavin to heme was identical to the steady-state reduction rate of cytochrome c in both WT and F166Y. These results clearly indicate that the first and second electrons of two-electron-reduced CDH are both transferred via heme, and that the redox reaction of CDH involves an electron-transfer chain mechanism in cytochrome c reduction.

Carbohydrate Dehydrogenases↗

PubMed2005

Characterization of carbohydrate-binding cytochrome b562 from the white-rot fungus Phanerochaete chrysosporium.

cDNA encoding a hemoprotein similar to the cytochrome domain of extracellular flavocytochrome cellobiose dehydrogenase (CDH) was cloned from the white-rot fungus Phanerochaete chrysosporium. The deduced amino acid sequence implies that there is a two-domain structure consisting of an N-terminal cytochrome domain and a C-terminal family 1 carbohydrate-binding module (CBM1) but that the flavin-containing domain of CDH is not present. The gene transcripts were observed in cultures in cellulose medium but not in cultures in glucose medium, suggesting that there is regulation by carbon catabolite repression. The gene was successfully overexpressed in Pichia pastoris, and the recombinant protein was designated carbohydrate-binding cytochrome b562 (CBCyt. b562). The resonance Raman spectrum suggested that the heme of CBCyt. b562 is 6-coordinated in both the ferric and ferrous states. Moreover, the redox potential measured by cyclic voltammetry was similar to that of the cytochrome domain of CDH. These results suggest that the redox characteristics may be similar to those of the cytochrome domain of CDH, and so CBCyt. b562 may have an electron transfer function. In a binding study with various carbohydrates, CBCyt. b562 was adsorbed with high affinity on both cellulose and chitin. As far as we know, this is the first example of a CBM1 connected to a domain without apparent catalytic activity for carbohydrate; this CBM1 may play a role in localization of the redox protein on the surface of cellulose or on the fungal sheath in vivo.

Amino Acid Sequence↗

PubMed2004

Structure and direct electrochemistry of cytochrome P450 from the thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7.

Cytochrome P450 from thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7 (P450st) has been expressed in Escherichia coli and purified at high homogeneity. P450st was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=53.6 A, b=55.1 A, and c=130.9 A, and the structure was determined at a 3.0 A resolution. The final R-factor was 0.194 (Rfree=0.235). Structural comparison with cytochrome P450 from S. solfataricus (CYP119) suggests that the region composed of the F to G helices and the Cl- binding site is responsible for the affinity for a ligand coordinating heme iron. Direct electrochemistry of P450st in a didodecyldimethylammonium bromide (DDAB) film on a plastic formed carbon (PFC) electrode has also been demonstrated. A quasi-reversible redox response has been observed even at elevated temperatures of up to 80 degrees C.

Bacterial Proteins↗

PubMed2002

Rates of oxygen and hydrogen exchange as indicators of TPQ cofactor orientation in amine oxidases.

This study presents the first detailed examination by resonance Raman (RR) spectroscopy of the rates of solvent exchange for the C5 and C3 positions of the TPQ cofactor in several wild-type copper-containing amine oxidases and mutants of the amine oxidase from Hansenula polymorpha (HPAO). On the basis of crystal structure analysis and differing rates of C5 [double bond] O and C3 [bond] H exchange within the enzyme systems, but equally rapid rates of C5 [double bond] O and C3 [bond] H exchange in a TPQ model compound, it is proposed that these data can be used to determine the TPQ cofactor orientation within the active site of the resting enzyme. A rapid rate of C5 [double bond] O exchange (t(1/2) < 30 min) and a slow (t(1/2) = 6 h) to nonexistent rate of C3 [bond] H exchange was observed for wild-type HPAO, the amine oxidase from Arthrobacter globiformis, pea seedling amine oxidase at pH 7.1, and the E406Q mutant of HPAO. This pattern is ascribed to a productive TPQ orientation, with the C5 [double bond] O near the substrate-binding site and the C3 [bond] H near the Cu. In contrast, a slow rate of C5 [double bond] O exchange (t(1/2) = 1.6-3.3 h) coupled with a fast rate of C3 [bond] H exchange (t(1/2) < 30 min) was observed for the D319E and D319N catalytic base mutants of HPAO and for PSAO at pH 4.6 (t(1/2) = 4.5 h for C5 [double bond] O exchange). This pattern identifies a flipped orientation, involving 180 degrees rotation about the C alpha-C beta bond, which locates the C3 [bond] H near the substrate-binding site and the C5 double bond] O near the Cu. Finally, fast rates of both C5 [double bond] O and C3 [bond] H exchange (t(1/2) < 30 min) were observed for the amine oxidase from Escherichia coli and the N404A mutant of HPAO, suggesting a mobile cofactor, with multiple TPQ orientations between productive and flipped. These results demonstrate that opposing sides of the TPQ ring possess different degrees of solvent accessibility and that the rates of C5 [double bond] O and C3 [bond] H exchange can be used to predict the TPQ cofactor orientation in the resting forms of these enzymes.

Amine Oxidase (Copper-Containing)↗

PubMedDate not supplied

Spectroscopic characterization and electrochemistry of poly(ethylene oxide)-modified myoglobin in organic solvents.

Myoglobin (Mb) was chemically modified with activated poly(ethylene oxide) (PEO) (average molecular weight of 2000) to solubilize it in various organic solvents. UV-vis, circular dichroism, and Raman spectroscopy were used to characterize the structure correlated with the electron-transfer reactions of PEO-modified Mb (PEO-Mb). Spectroscopic data indicated changes in heme coordination geometry for PEO-Mb in various organic solvents that are different from that in water. The Raman spectrum showed the characteristics of PEO-Mb in PEO oligomer (average MW of 200) in the five-coordinate high-spin state, which facilitates fast electron-transfer reactions between protein and the glassy carbon electrode. These results suggest heme environment effects on the properties of proteins in organic solvents.

Animals↗