Bionanotube tetrapod assembly by in situ synthesis of a gold nanocluster with (Gp5-His6)3 from bacteriophage T4.
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Bacteriophage T4↗
Biomedical subjects
Takafumi Ueno
Publications and source records attributed to Takafumi Ueno.
Design of metal cofactors activated by a protein-protein electron transfer system.
Protein-to-protein electron transfer (ET) is a critical process in biological chemistry for which fundamental understanding is expected to provide a wealth of applications in biotechnology. Investigations of protein-protein ET systems in reductive activation of artificial cofactors introduced into proteins remains particularly challenging because of the complexity of interactions between the cofactor and the system contributing to ET. In this work, we construct an artificial protein-protein ET system, using heme oxygenase (HO), which is known to catalyze the conversion of heme to biliverdin. HO uses electrons provided from NADPH/cytochrome P450 reductase (CPR) through protein-protein complex formation during the enzymatic reaction. We report that a Fe(III)(Schiff-base), in the place of the active-site heme prosthetic group of HO, can be reduced by NADPH/CPR. The crystal structure of the Fe(10-CH(2)CH(2)COOH-Schiff-base).HO composite indicates the presence of a hydrogen bond between the propionic acid carboxyl group and Arg-177 of HO. Furthermore, the ET rate from NADPH/CPR to the composite is 3.5-fold faster than that of Fe(Schiff-base).HO, although the redox potential of Fe(10-CH(2)CH(2)COOH-Schiff-base).HO (-79 mV vs. NHE) is lower than that of Fe(Schiff-base).HO (+15 mV vs. NHE), where NHE is normal hydrogen electrode. This work describes a synthetic metal complex activated by means of a protein-protein ET system, which has not previously been reported. Moreover, the result suggests the importance of the hydrogen bond for the ET reaction of HO. Our Fe(Schiff-base).HO composite model system may provide insights with regard to design of ET biosystems for sensors, catalysts, and electronics devices.
Binding Sites↗
Coordinated design of cofactor and active site structures in development of new protein catalysts.
New methods for the synthesis of artificial metalloenzymes are important for the construction of novel biocatalysts and biomaterials. Recently, we reported new methodology for the synthesis of artificial metalloenzymes by reconstituting apo-myoglobin with metal complexes (Ohashi, M. et al., Angew Chem., Int. Ed. 2003, 42, 1005-1008). However, it has been difficult to improve their reactivity, since their crystal structures were not available. In this article, we report the crystal structures of M(III)(Schiff base).apo-A71GMbs (M = Cr and Mn). The structures suggest that the position of the metal complex in apo-Mb is regulated by (i) noncovalent interaction between the ligand and surrounding peptides and (ii) the ligation of the metal ion to proximal histidine (His93). In addition, it is proposed that specific interactions of Ile107 with 3- and 3'-substituent groups on the salen ligand control the location of the Schiff base ligand in the active site. On the basis of these results, we have successfully controlled the enantioselectivity in the sulfoxidation of thioanisole by changing the size of substituents at the 3 and 3' positions. This is the first example of an enantioselective enzymatic reaction regulated by the design of metal complex in the protein active site.
Apoproteins↗
Monooxygenation of an aromatic ring by F43W/H64D/V68I myoglobin mutant and hydrogen peroxide. Myoglobin mutants as a model for P450 hydroxylation chemistry.
Myoglobin (Mb) is used as a model system for other heme proteins and the reactions they catalyze. The latest novel function to be proposed for myoglobin is a P450 type hydroxylation activity of aromatic carbons (Watanabe, Y., and Ueno, T. (2003) Bull. Chem. Soc. Jpn. 76, 1309-1322). Because Mb does not contain a specific substrate binding site for aromatic compounds near the heme, an engineered tryptophan in the heme pocket was used to model P450 hydroxylation of aromatic compounds. The monooxygenation product was not previously isolated because of rapid subsequent oxidation steps (Hara, I., Ueno, T., Ozaki, S., Itoh, S., Lee, K., Ueyama, N., and Watanabe, Y. (2001) J. Biol. Chem. 276, 36067-36070). In this work, a Mb variant (F43W/H64D/V68I) is used to characterize the monooxygenated intermediate. A modified (+16 Da) species forms upon the addition of 1 eq of H2O2. This product was digested with chymotrypsin, and the modified peptide fragments were isolated and characterized as 6-hydroxytryptophan using matrix-assisted laser desorption ionization time-of-flight tandem mass spectroscopy and 1H NMR. This engineered Mb variant represents the first enzyme to preferentially hydroxylate the indole side chain of Trp at the C6 position. Finally, heme extraction was used to demonstrate that both the formation of the 6-hydroxytryptophan intermediate (+16 Da) and subsequent oxidation to form the +30 Da final product are catalyzed by the heme cofactor, most probably via the compound I intermediate. These results provide insight into the mechanism of hydroxylation of aromatic carbons by heme proteins, demonstrating that non-thiolate-ligated heme enzymes can perform this function. This establishes Mb compound I as a model for P450 type aromatic hydroxylation chemistry.
5-Hydroxytryptophan↗
A virus-based nanoblock with tunable electrostatic properties.
Five different "HIS tag" mutants of cowpea mosaic virus were made by genetically introducing six contiguous histidine residues at various locations on the virus capsid. The mutant particles showed differential affinity for binding nickel, and their electrostatic properties could be controlled as a function of the protonation state of the exposed histidine sequence. The specific addressability of the HIS tag was corroborated by the selective modification of the histidine sequence with nanogold cross-linked to the Ni-NTA moiety.
Amino Acid Sequence↗
Catalase reaction by myoglobin mutants and native catalase: mechanistic investigation by kinetic isotope effect.
The catalase reaction has been studied in detail by using myoglobin (Mb) mutants. Compound I of Mb mutants (Mb-I), a ferryl species (Fe(IV)=O) paired with a porphyrin radical cation, is readily prepared by the reaction with a nearly stoichiometric amount of m-chloroperbenzoic acid. Upon the addition of H2O2 to an Mb-I solution, Mb-I is reduced back to the ferric state without forming any intermediates. This indicates that Mb-I is capable of performing two-electron oxidation of H2O2 (catalatic reaction). Gas chromatography-mass spectroscopy analysis of the evolved O2 from a 50:50 mixture of H2(18)O2/H2(16)O2 solution containing H64D or F43H/H64L Mb showed the formation of 18O2 (m/e = 36) and 16O2 (m/e = 32) but not 16O18O (m/e = 34). This implies that O2 is formed by two-electron oxidation of H2O2 without breaking the O-O bond. Deuterium isotope effects on the catalatic reactions of Mb mutants and catalase suggest that the catalatic reactions of Micrococcus lysodeikticus catalase and F43H/H64L Mb proceed via an ionic mechanism with a small isotope effect of less than 4.0, since the distal histidine residue is located at a proper position to act as a general acid-base catalyst for the ionic reaction. In contrast, other Mb mutants such as H64X (X is Ala, Ser, and Asp) and L29H/H64L Mb oxidize H2O2 via a radical mechanism in which a hydrogen atom is abstracted by Mb-I with a large isotope effect in a range of 10-29, due to a lack of the general acid-base catalyst.
Animals↗
Size-selective olefin hydrogenation by a Pd nanocluster provided in an apo-ferritin cage.
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Journal Article↗
Crystal structures of artificial metalloproteins: tight binding of FeIII(Schiff-Base) by mutation of Ala71 to Gly in apo-myoglobin.
Apo-myoglobin (apo-Mb) and apo-A71GMb were successfully reconstituted with FeIII(salophen) (1) (salophen = N,N'-bis(salicylidene)-1,2-phenilenediamine), Fe(III)(3,3'-Me2-salophen) (2), and FeIII(5,5'-t-Bu2-salophen) (3). The crystal structure of 2.apo-A71GMb shows the tight binding of the complex in the Mb cavity, while in wild-type apo-Mb it is highly disordered due to the steric repulsion with Ala71. Furthermore, the structure of 2.apo-A71GMb suggests a possible accommodation of a small substrate in the cavity. In fact, the cyanide association rate constant of 2.apo-A71GMb is 216-fold larger compared to that of 2.apo-Mb. These results provide us principles for the noncovalent fixation of synthetic metal cofactors at the desired positions in protein matrixes.
Alanine↗
Copper binding to plant ozone-inducible proteins (OI2-2 and OI14-3).
Ozone-inducible proteins (OI2-2 and OI14-3) from Atriplex canescens whose structure and function are unknown are rich in glycine intercepted with histidine and tyrosine with putative signal peptides at the N-terminus. OI2-2 and OI14-3 contain 8 and 10 tandem repeats of YGHGGG, respectively. In order to study whether these proteins bind Cu(2+), circular dichroism (CD), and nuclear magnetic resonance (NMR) were measured for four synthetic peptides corresponding to sections of the sequences of these proteins; 1 (HGGGY), 2 (HGGGYGH), 3 (YGHGGGY), and 4 (YGHGGGYGHGGGY), where all peptides were chemically blocked with an acetyl group at the N-terminus and an -NH(2) group at the C-terminus. Visible CD spectra of the four peptides show positive peaks near 580 and 340nm, which were observed at pH 7.4 but not pH 6.0, indicating clearly that the four peptides bind Cu(2+). The NMR spectra indicate that the addition of small amounts of CuSO(4) to 3 (Y1-G2-H3-G4-G5-G6-Y7) causes significant broadening of resonances of the side chain protons (C(beta)H, C(epsilon1)H, and C(delta2)H) of His3 and the side chain C(beta)H of Tyr1 at pH 7.4. In addition, the backbone C(alpha)H resonances of Gly2 and Gly4 were broadened more strongly than those of Gly5 and Gly6. CD titration experiment suggested that two repeats of YGHGGG comprise the fundamental Cu(2+) binding unit. Thus, the ozone-inducible proteins are capable of binding at least four or five copper ions per protein. These copper-binding proteins would function as active oxygen scavengers.
Amino Acid Sequence↗
Hybridization of modified-heme reconstitution and distal histidine mutation to functionalize sperm whale myoglobin.
To modulate the physiological function of a hemoprotein, most approaches have been demonstrated by site-directed mutagenesis. Replacement of the native heme with an artificial prosthetic group is another way to modify a hemoprotein. However, an alternate method, mutation or heme reconstitution, does not always demonstrate sufficient improvement compared with the native heme enzyme. In the present study, to convert a simple oxygen storage hemoprotein, myoglobin, into an active peroxidase, we applied both methods at the same time. The native heme of myoglobin was replaced with a chemically modified heme 2 having two aromatic rings at the heme-propionate termini. The constructed myoglobins were examined for 2-methoxyphenol (guaiacol) oxidation in the presence of H2O2. Compared with native myoglobin, rMb(H64D.2) showed a 430-fold higher kcat/Km value, which is significantly higher than that of cytochrome c peroxidase and only 3-fold less than that of horseradish peroxidase. In addition, myoglobin-catalyzed degradation of bisphenol A was examined by HPLC analysis. The rMb(H64D.2) showed drastic acceleration (>35-fold) of bisphenol A degradation compared with the native myoglobin. In this system, a highly oxidized heme reactive species is smoothly generated and a substrate is effectively bound in the heme pocket, while native myoglobin only reversibly binds dioxygen. The present results indicate that the combination of a modified-heme reconstitution and an amino acid mutation should offer interesting perspectives toward developing a useful biomolecule catalyst from a hemoprotein.
Animals↗
Molecular engineering of myoglobin: influence of residue 68 on the rate and the enantioselectivity of oxidation reactions catalyzed by H64D/V68X myoglobin.
In the elucidation of structural requirements of heme vicinity for hydrogen peroxide activation, we found that the replacement of His-64 of myoglobin (Mb) with a negatively charged aspartate residue enhanced peroxidase and peroxygenase activities by 78- and 580-fold, respectively. Since residue 68 is known to influence the ligation of small molecules to the heme iron, we constructed H64D/V68X Mb bearing Ala, Ser, Leu, Ile, and Phe at position 68 to improve the oxidation activity. The Val-68 to Leu mutation of H64D Mb accelerates the reaction with H(2)O(2) to form a catalytic species, called compound I, and improves the one-electron oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (i.e., peroxidase activity) approximately 2-fold. On the other hand, H64D/V68I Mb oxygenates thioanisole 2.7- and 1600-fold faster than H64D and wild-type Mb, respectively. In terms of the enantioselectivity, H64D/V68A and H64D/V68S Mb were good chiral catalysts for thioanisole oxidation and produced the (R)-sulfoxide dominantly with 84% and 88% ee, respectively [Kato, S., et al. (2002) J. Am. Chem. Soc. 124, 8506-8507]. On the contrary, the substitution of Val-68 in H64D Mb with an isoleucine residue alters the dominant sulfoxide product from the (R)- to the (S)-isomer. The crystal structures of H64D/V68A and H64D/V68S Mb elucidated in this study do not clearly indicate residues interacting with thioanisole. However, comparison of the active site structures provides the basis to interpret the changes in oxidation activity: (1) direct steric interactions between residue 68 and substrates (i.e., H(2)O(2), ABTS, thioanisole) and (2) the polar interactions between tightly hydrogen-bonded water molecules and substrates.
Amino Acid Substitution↗
Preparation of artificial metalloenzymes by insertion of chromium(III) Schiff base complexes into apomyoglobin mutants.
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Apoproteins↗
Vasomotor function of pig coronary arteries after chronic coronary occlusion.
Placement of an ameroid constrictor in large-conduit pig coronary arteries causes progressive stenosis and distal myocardial ischemia. Blood perfusion in the ischemic region is partly dependent on vasomotor responses to neural and humoral factors distal to the occlusion site. To ascertain the degree of impairment of vascular function in pigs, the authors induced myocardial ischemia by placing an ameroid constrictor in the left circumflex coronary artery and examined vascular reactivity and histopathology distal to the constriction site. The sensitivity of the distal left circumflex coronary and nonoccluded control left anterior descending arteries to PGF(2alpha) was similar. After nitric oxide blockade using Nw-nitro-l-arginine methylester (l-NAME), the sensitivity and maximal contraction to PGF(2alpha) were significantly increased in both the left circumflex coronary (EC50: 5.86 +/- 0.74 vs. 3.28 +/- 0.84 microM; C(max): 4.63 +/- 0.28 vs. 6.25 +/- 0.30 g, P < 0.01) and left anterior descending (EC50: 6.57 +/- 0.73 vs. 2.78 +/- 0.16 microM; C(max): 5.09 +/- 0.37 vs. 6.95 +/- 0.39 g, P < 0.01) arteries. Substance P-induced relaxation (100 pM) was blocked to a larger degree in the distal left circumflex coronary artery when compared with the left anterior descending artery (76.9 +/- 4.2% vs. 56.4 +/- 3.1%, P < 0.05). Endothelium-independent relaxation to sodium nitroprusside was similar in the left circumflex coronary and left anterior descending arteries before and after nitric oxide blockade. Histopathologic examination showed no major differences between distal left circumflex coronary artery segments and left anterior descending artery controls. However, scanning electron microscopy showed endothelial hypertrophy and activation in specimens from the left circumflex coronary arteries. In summary, as a result of the major hemodynamic changes induced by a chronic constriction and eventual occlusion of a large coronary artery, distal segments underwent adaptive compensatory changes. Such compensation may be related to an increased nitric oxide production by the hypertrophic endothelium in response to alterations in coronary hemodynamics.
Animals↗
[Diagnostic criteria for syndrome X].
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Humans↗
Asymmetric sulfoxidation and amine binding by H64D/V68A and H64D/V68S Mb: mechanistic insight into the chiral discrimination step.
The H64D/V68A and H64D/V68S mutants of Myoglobin are found to oxidize thioanisole with high enantioselectivity and reactivity. These mutants are also capable of enantioselective binding of alpha-methylbenzylamine, which mimics an expected sulfoxidation intermediate. The kinetic study of the amine binding shows that the Fe-O bond cleavage in the intermediate may be the chiral discrimination step of the sulfoxidation.
Kinetics↗
Sequential follow-up results of catheter intervention for coronary artery lesions after Kawasaki disease: quantitative coronary artery angiography and intravascular ultrasound imaging study.
BACKGROUND: The purpose of this study was to assess the sequential follow-up results of catheter intervention in Kawasaki disease by use of quantitative coronary angiography (QCA) and intravascular ultrasound imaging. METHODS AND RESULTS: Catheter intervention was performed on 23 stenotic lesions in 22 patients (aged 2 to 24 years). Percutaneous balloon angioplasty (PBA) was performed in 4 patients, stent implantation in 7, percutaneous transluminal coronary rotational ablation (PTCRA) in 10, and a combination of PTCRA with stent implantation in 2. A total of 21 lesions (91%) were successfully dilated by catheter intervention without major or minor complications. One patient immediately underwent coronary artery bypass grafting (CABG) surgery because stent implantation failed to resolve his lesion. At 4 to 6 months after catheter intervention, 2 restenotic lesions (9%) were detected by QCA in 2 patients who had undergone PBA, and these patients subsequently underwent CABG surgery. In 6 months to 3 years after catheter intervention, no patients showed evidence of ischemic findings. At 3 to 4 years after catheter intervention, QCA and intravascular ultrasound studies were performed on 15 lesions in 14 patients. Two restenotic lesions (13%) were detected by QCA in 2 patients. One of the 2 had stent implantation and underwent CABG surgery, and the other had undergone PTCRA and underwent re-PTCRA. Thirteen patients demonstrated no ischemic findings at 3 to 8 years after catheter intervention. CONCLUSION: Catheter intervention for Kawasaki disease can be accomplished and can be effective in the short term, but the long-term efficacy should be verified by further study.
Adolescent↗
Coexistence of impairment of endothelium-derived nitric oxide and platelet-derived nitric oxide in patients with coronary risk factors.
Impairment of endothelium-derived nitric oxide (EDNO) has been demonstrated in patients with coronary risk factors in some studies, as well as impaired platelet-derived nitric oxide (PDNO) in other studies. However, no study has examined whether these impairments coexist. In 24 patients with coronary risk factors, femoral vascular endothelial function was assessed with acetylcholine (ACh: 50, 100, 200 and 400 microg/min) and endothelium-independent vascular function with nitroglycerin (NTG; 50, 100, 200 microg/min) using a Doppler flow-wire technique, as well as ADP (5 micromol/L)-induced PDNO release with an NO-specific electrode. The ACh-mediated percent change in femoral vascular resistance index (% change of FVRI) and PDNO release had a significant correlation with the number of risk factors. The ACh-mediated % change of FVRI, but not that with NTG, significantly correlated with the PDNO release. Both EDNO and PDNO bioactivities are impaired in patients with coronary risk factors and there is a common mechanism.
Acetylcholine↗
Role of the Invariant Peptide Fragment Forming NH.S Hydrogen Bonds in the Active Site of Cytochrome P-450 and Chloroperoxidase: Synthesis and Properties of Cys-Containing Peptide Fe(III) and Ga(III) (Octaethylporphinato) Complexes as Models.
The primary sequence of Cys-X-Gly-Y- (X, hydrophobic residue; Y, hydrophilic residue) is highly conserved in cytochrome P-450s. The amide NHs of Leu, Gly, and X are assumed to form NH.S hydrogen bonds which are also found in the active site fragment, Cys-Pro-Ala-Leu, of chloroperoxidase (CPO). [Fe(III)(OEP)(Z-cys-Leu-Gly-Leu-OMe)] (OEP, octaethylporphinato; Z, benzyloxycarbonyl) and [Fe(III)(OEP)(Z-cys-Pro-Ala-Leu-OMe)] were synthesized as P-450 and CPO model complexes containing the invariant amino acid fragment of the active site. The corresponding gallium(III) complexes were also synthesized to investigate the solution structures using two-dimensional (2D) NMR experiments because the Ga(III) ion is similar to the Fe(III) ion in the ionic radii and in the coordination geometry. The solution structures of the peptide part of the gallium complexes indicate that the invariant fragments maintain a beta I-turn-like conformation and then form NH.S hydrogen bonds between S(gamma)Cys and NH of the third and fourth amino acid residues. The hydrogen bonds have also been confirmed by the (2)H NMR spectra of N(2)H-substituted Fe(III) peptide complexes. The Fe(III)/Fe(II) redox potentials of the Fe(III) complexes indicate that the NH.S hydrogen bonds in the fragments causes a slight positive shift of the redox potential. The tri- and tetrapeptide Fe(III) complexes containing the invariant fragments of P-450 are kinetically stable at 30 degrees C in CH(2)Cl(2). In contrast, [Fe(III)(OEP)(Z-cys-Leu-OMe)] decomposed to give [Fe(II)(OEP)] (22%) and the corresponding disulfide immediately in CD(2)Cl(2) at 30 degrees C for 1 h. These results indicate that the invariant fragments involving the hydrogen bonds cause the stabilization of the high-spin Fe(III) resting state rather than the positive shift of Fe(III)/Fe(II) redox potential.
Journal Article↗