Biomedical subjects

Y Anraku

Publications and source records attributed to Y Anraku.

At least 73 records · Page 4Linked to original sources

PubMed1994

Observation of the Fe-O2 and FeIV=O stretching Raman bands for dioxygen reduction intermediates of cytochrome bo isolated from Escherichia coli.

Reaction intermediates in dioxygen reduction by the E. coli cytochrome bo-type ubiquinol oxidase were studied by time-resolved resonance Raman spectroscopy using the artificial cardiovascular system. At 0-20 microseconds following photolysis of the enzyme-CO adduct in the presence of O2, we observed the Fe-O2 stretching Raman band at 568 cm-1 which shifted to 535 cm-1 with the 18O2 derivative. These frequencies are remarkably close to those of other oxyhemoproteins including dioxygen-bound hemoglobin and aa3-type cytochrome c oxidase. In the later time range (20-40 microseconds), other oxygen-isotope-sensitive Raman bands were observed at 788 and 361 cm-1. Since the 781 cm-1 band exhibited a downshift by 37 cm-1 upon 18O2 substitution, we assigned it to the FeIV=O stretching mode. This band is considered to arise from the ferryl intermediate, but its appearance was much earlier than the corresponding intermediate of bovine cytochrome c oxidase (> 100 microseconds). The 361 cm-1 band showed the 16O/18O isotopic frequency shift of 14 cm-1 similar to the case of bovine cytochrome c oxidase reaction.

Cytochrome b Group↗

PubMed1994

An Escherichia coli cyoE gene homologue in thermophilic Bacillus PS3 encodes a thermotolerant heme O synthase.

The cyoE gene of the Escherichia coli bo-type quinol oxidase operon (cyoABCDE) has been previously shown to encode heme O synthase. To demonstrate a catalytic role of a cyoE homologue (the caaE gene) in the gene cluster for caa3-type cytochrome c oxidase of thermophilic Bacillus PS3, we have carried out genetic complementation analysis using the chimeric operon cyoABCD-caaE and heme O synthase assay using the CaaE-overproduced E. coli membranes. We found that the caaE gene encodes a thermotolerant heme O synthase which provides an intermediate for heme A biosynthesis.

Alkyl and Aryl Transferases↗

PubMed1994

Resonance Raman and Fourier transform infrared studies on the subunit I histidine mutants of the cytochrome bo complex in Escherichia coli. Molecular structure of redox metal centers.

The cytochrome bo complex is a heme-copper terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. Site-directed mutagenesis studies were greatly successful in identifying ligands of the redox centers in the oxidase (Minagawa, J., Mogi, T., Gennis, R. B., and Anraku, Y. (1992) J. Biol. Chem. 267, 2096-2104). In the present study, resonance Raman and Fourier transform infrared spectroscopies were applied to the subunit I histidine mutant oxidases, and the active-site structure was studied in detail. In the dithionite-reduced state, the wild-type oxidase and the His-284-->Ala and His-333-->Ala mutant oxidases showed two Raman lines (v3) at approximately 1492 and 1472 cm-1, which are attributable to low- and high-spin heme components, respectively. The Ala substitutions at His-106 and His-421 reduced the low-spin line, whereas the His-419-->Ala mutation eliminated the high-spin line. This indicates that His-106 and His-421 are the axial ligands of the low-spin heme B, while His-419 acts as a proximal ligand of the high-spin heme O. The v(Fe-CO) stretching frequency in the wild-type oxidase down-shifted from 523 to 498 cm-1 in the CuB-deficient mutant oxidase (His-333-->Ala), while the v(C-O) stretching frequency up-shifted from 1960 to 1970 cm-1. These frequency differences were ascribed to the contribution from the reduced CuB at the binuclear center. It may indicate that delocalization of electrons at the CuB center toward the heme O iron is facilitated by a bridge ligand structure at the binuclear center and is essential for driving dioxygen reduction chemistry, and therefore, redox-coupled proton pumping.

Alanine↗

PubMed1994

Site-specific alteration of arginine 376, the unique positively charged amino acid residue in the mid-membrane-spanning regions of the proline carrier of Escherichia coli.

An alignment of 5 amino acids in the Escherichia coli proline carrier (G328-A366-L371-GR376) is common in the amino acid sequences of several Na+ symport carriers, and it has been proposed as the putative sodium binding motif (Deguchi, Y., Yamato, I., and Anraku, Y. (1990) J. Biol. Chem. 265, 21704-21708). To determine whether these amino acids are essential for Na+ symport activity as the Na+ binding site, one of the amino acids in this alignment, Arg-376, which is the only positively charged amino acid in the innermost part of the predicted membrane-spanning regions, was changed to either lysine, glutamine, or glutamic acid by oligonucleotide dependent site-specific mutagenesis. The transport and binding activities of the proline of the R376K mutant carrier were not detected at all. The activities of the other mutant carriers for uptake and binding of proline were as high as those of the wild-type carrier. These two mutant carriers were as sensitive to the proline analogue azetidine-2-carboxylate and to N-ethylmaleimide as the wild-type carrier, indicating that they have the same properties as the wild-type. The amounts of the carrier proteins expressed from these mutated putP genes were similar to that from the wild-type gene. These results imply that the Arg-376 in the proline carrier does not reside at the sodium binding site, suggesting that the similar alignment found in the amino acid sequences of several Na+ symport carriers is not essential for the transport or binding activities, although this similar alignment may have some relevance to the structure of the Na+ symporter. Furthermore, that the only Arg residue in the middle part of the predicted membrane-spanning regions is dispensable for the energy coupling activity indicates a unique difference of the coupling mechanism from the other secondary active transport systems, such as that of the lactose permease and the tetracycline/H+ antiporter.

Amino Acid Sequence↗

PubMed1994

A novel gene, STT4, encodes a phosphatidylinositol 4-kinase in the PKC1 protein kinase pathway of Saccharomyces cerevisiae.

A staurosporine-sensitive mutation (stt1) in yeast has been found in the PKC1 gene that encodes a protein kinase C homologue (Yoshida, S., Ikeda, E., Uno, I., and Mitsuzawa, H. (1992) Mol. Gen. Genet. 231, 337-344). We report here another staurosporine-sensitive mutant, stt4, which shows very similar phenotypes to that of the stt1 mutant. The stt4 temperature-sensitive mutant arrests mostly in G2/M phase at 37 degrees C, and the stt4 deletion mutant shows an osmoremedial phenotype. Staurosporine sensitivity of the stt4 mutant was suppressed by overexpression of PKC1/STT1, indicating genetic interaction between stt4 and pkc1/stt1. The nucleotide sequence of STT4 predicts a hydrophilic protein composed of 1,900 amino acid residues, with 26% sequence identity to the yeast VPS34 gene product and 27% to the catalytic subunit of mammalian phosphatidylinositol (PI) 3-kinase, respectively. Cell homogenates of the stt4 deletion mutant show normal PI3-kinase activity but lack most of the PI4-kinase activity that is detected in the wild-type. We conclude that STT4 encodes a yeast PI4-kinase that functions in the PKC1 protein kinase pathway.

1-Phosphatidylinositol 4-Kinase↗

PubMed1994

Genetic interaction between the Ras-cAMP pathway and the Dis2s1/Glc7 protein phosphatase in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae DIS2S1/GLC7 gene encodes a type 1 protein phosphatase indispensable for cell proliferation. We found that introduction of a multicopy DIS2S1 plasmid impaired growth of cells with reduced activity of the cAMP-dependent protein kinase. In order to understand further the interaction between the two enzymes, a temperature-sensitive mutation in the DIS2S1 gene was isolated. The mutant accumulated less glycogen than wild type at the permissive temperature, indicating that activity of the Dis2s1 protein phosphatase is attenuated by the mutation. Furthermore, the dis2s1ts mutation was shown to be suppressed by a multicopy plasmid harboring PDE2, a gene for cAMP phosphodiesterase. These results indicate that the Ras-cAMP pathway interacts genetically with the DIS2S1/GLC7 gene.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1994

Genetic interactions among genes involved in the STT4-PKC1 pathway of Saccharomyces cerevisiae.

Loss of yeast protein kinase C function results in three distinct phenotypes: staurosporine sensitivity, cell lysis and blockage of cell cycle progression at the G2/M boundary. Genetic analysis of the PKC1/STT1 protein kinase C gene and its interactions with STT4, encoding an upstream phosphatidylinositol 4-kinase, and BCK1, encoding a downstream protein kinase, reveal that they form part of a single pathway. However, the BCK1-20 mutation (a gain-of-function mutation of BCK1) or overexpression of PKC1 cannot suppress all of the phenotypes caused by the loss of STT4 function, strongly suggesting the existence of a branch point between STT4 and PKC1. We also describe the MSS4 gene, a multicopy suppressor of the temperature-sensitive stt4-1 mutation. MSS4 is predicted to encode a hydrophilic protein of 779 amino acid residues and is essential for cell growth. Based on genetic and biochemical data, we suggest that MSS4 acts downstream of STT4, but in a pathway that does not involve PKC1.

1-Phosphatidylinositol 4-Kinase↗

PubMed1994

Chemiosmotic coupling of ion transport in the yeast vacuole: its role in acidification inside organelles.

Acidification inside the vacuo-lysosome systems is ubiquitous in eukaryotic organisms and essential for organelle functions. The acidification of these organelles is accomplished by proton-translocating ATPase belonging to the V-type H(+)-ATPase superfamily. However, in terms of chemiosmotic energy transduction, electrogenic proton pumping alone is not sufficient to establish and maintain those compartments inside acidic. Current studies have shown that the in situ acidification depends upon the activity of V-ATPase and vacuolar anion conductance; the latter is required for shunting a membrane potential (interior positive) generated by the positively charged proton translocation. Yeast vacuoles possess two distinct Cl- transport systems both participating in the acidification inside the vacuole, a large acidic compartment with digestive and storage functions. These two transport systems have distinct characteristics for their kinetics of Cl- uptake or sensitivity to a stilbene derivative. One shows linear dependence on a Cl- concentration and is inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). The other shows saturable kinetics with an apparent Km for Cl- of approximately 20 mM. Molecular mechanisms of the chemiosmotic coupling in the vacuolar ion transport and acidification inside are discussed in detail.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1994

Protozyme: emerging evidence in nature.

The VMA1 locus in Saccharomyces cerevisiae contains a nested genetic element, the VDE gene, and expresses two functional proteins. A single VMA1 translational product seems to catalyze a self protein splicing in which an internal domain is excised out to produce a site-specific DNA endonuclease and the N- and C-domains are ligated by a transpeptidation reaction to yield a catalytic subunit of the vacuolar ATPase. Accumulating evidence in the past few years suggests that the VMA1 locus encodes a protozyme (for protos en zyme) which has dual roles in life as a protean catalyst for self protein splicing and for self gene homing. Four protozymes that share a common mechanism in protein splicing have been found in four organisms covering three major phylogenic trees.

Amino Acid Sequence↗

PubMed1994

Molecular structure of a heme-copper redox center of the Escherichia coli ubiquinol oxidase: evidence and model.

Biochemical, spectroscopic, and molecular biological studies on bacterial respiratory oxidases in the last decade have greatly increased our understanding of a molecular structure of the metal centers which catalyze the dioxygen reduction chemistry. Based upon the latest physicochemical and molecular biological evidence and theoretical consideration of a folding mechanics of membrane proteins, we propose here the tertiary structure of the heme-copper metal center of the Escherichia coli bo-type ubiquinol oxidase, the cytochrome bo complex. The molecular mechanism of electron transfer-coupled proton pumping in the heme-copper respiratory oxidases is reviewed on the basis of this predicted model.

Amino Acid Sequence↗

PubMed1994

Biosynthesis and functional role of haem O and haem A.

Haem O and/or haem A are specifically synthesized for the haem-copper respiratory oxidases. A 17-carbon hydroxyethylfarnesyl chain at the pyrrole ring A of the haems seems essential for catalytic functions at the oxygen-reduction site. The discovery of haem O in the cytochrome bo complex from Escherichia coli was a breakthrough in the studies on haem A biosynthesis. Molecular biological and biochemical studies in the past three years demonstrated that the cyoE/ctaB/COX10 genes are indispensable for functional expression of the terminal oxidases and encode a novel enzyme haem O synthase (protohaem IX farnesyltransferase). It has recently been suggested that the ctaA gene adjacent to the ctaB-ctaCDEF gene cluster in Bacillus subtilis encodes haem A synthase (haem O monooxygenase). In this article, we review current knowledge of the genes for haem O and haem A biosyntheses, the location and regulation of haem O synthase, the possible enzymatic mechanism of farnesyl transfer to haem B and the possible roles of the farnesylated haems.

Alkyl and Aryl Transferases↗

PubMed1994

MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca2+ influx and mating.

By establishing a unique screening method, we have isolated yeast mutants that die only after differentiating into cells with a mating projection, and some of them are also defective in Ca2+ signaling. The mutants were classified into five complementation groups, one of which we studied extensively. This mutation defines a new gene, designated MID1, which encodes an N-glycosylated, integral plasma membrane protein with 548 amino acid residues. The mid1-1 mutant has low Ca2+ uptake activity, loses viability after receiving mating pheromones, and escapes death when incubated with high concentrations of CaCl2. The MID1 gene is nonessential for vegetative growth. The efficiency of mating between MATa mid1-1 and MAT alpha mid1-1 cells is low. These results demonstrate that MID1 is required for Ca2+ influx and mating.

Amino Acid Sequence↗

PubMed1993

Identification of the functional domains in heme O synthase. Site-directed mutagenesis studies on the cyoE gene of the cytochrome bo operon in Escherichia coli.

The cytochrome bo complex is a terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and is encoded by the cyoABCDE operon. Recently, we have demonstrated that heme O at the high-spin heme-binding site is essential for redox-coupled proton pumping by the oxidase and suggested that the cyoE gene encodes a novel enzyme for heme O biosynthesis, protoheme IX farnesyltransferase (heme O synthase) (Saiki, K., Mogi, T., and Anraku, Y. (1992) Biochem. Biophys. Res. Commun. 189, 1491-1497). This study was focused to define the catalytic domain(s) of the CyoE protein via a site-directed mutagenesis approach. We have individually substituted 40 amino acid residues including 22 invariant residues with alanines and found that 23 mutant oxidases were nonfunctional and exhibited a specific loss of the CO binding activity at the site of the high-spin heme. Characterizations of the purified D65A, Y120A, and W172A mutant oxidases, which represent the mutations of different topological domains, revealed that their defects are attributable to substitution of protoheme IX for heme O present in the high-spin heme-binding site. Based on the above observations, we suggest that the conserved amino acid residues present in the cytoplasmic loops II/III and IV/V are part of the catalytic center of heme O synthase.

Alkyl and Aryl Transferases↗

PubMed1993

In vitro heme O synthesis by the cyoE gene product from Escherichia coli.

The cytochrome bo complex is a heme-copper terminal quinol oxidase in the aerobic respiratory chain of Escherichia coli and contains low spin heme B, high spin heme O and CuB as the redox metal centers in subunit I. Based on site-directed mutagenesis studies on the cyoE gene in the cytochrome bo operon, we have postulated that the cyoE gene encodes a protoheme IX farnesyltransferase (heme O synthase) (Saiki, K., Mogi, T., and Anraku, Y. (1992) Biochem. Biophys. Res. Commun. 189, 1491-1497). The present study demonstrates that the CyoE protein is localized in the cytoplasmic membrane and that the CyoE-overproduced membranes efficiently catalyze a conversion of exogenous ferrous protoheme IX and farnesyl diphosphate to heme O in the presence of divalent cations such as Mg2+ or Ca2+. Thus, the cyoABCDE operon in E. coli encodes not only subunits of the cytochrome bo complex but also heme O synthase that is specifically required for functional expression of the bo-type quinol oxidase. Heme O seems to be an intermediate in heme A biosynthesis.

Alkyl and Aryl Transferases↗

PubMed1993

Cytochrome d axial ligand of the bd-type terminal quinol oxidase from Escherichia coli.

Using various spectroscopic techniques, we studied the structure of the dioxygen reduction site of the bd-type terminal quinol oxidase in the aerobic respiratory chain of Escherichia coli. Resonance Raman and FT-IR spectroscopies identified the v(Fe(2+)-CO) and v(C-O) stretching frequencies at 471 and 1980.7 cm-1, respectively, at the cytochrome d center of the dithionite-reduced CO-bound enzyme. The CO ligation in the cytochrome bd complex is considerably different from those of the heme-copper terminal oxidases. Anaerobic addition of NO to the air-oxidized enzyme caused an exchange of cytochrome d-bound dioxygen with NO leading to an appearance of cytochrome d-NO EPR signal. But there is no superhyperfine structure originating from the cytochrome d proximal 14N ligand in the central resonance of the NO EPR signal. These results suggest that cytochrome d axial ligand of the cytochrome bd complex is likely a histidine residue in an anomalous condition or other than a histidine residue and, therefore, the molecular structure around the dioxygen-binding site is different from that of the heme-copper terminal oxidases.

Binding Sites↗

PubMed1993

VMA13 encodes a 54-kDa vacuolar H(+)-ATPase subunit required for activity but not assembly of the enzyme complex in Saccharomyces cerevisiae.

Previous purifications and characterizations of the Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) have indicated that this enzyme is a multisubunit complex composed of at least eight subunits of 100-, 69-, 60-, 42-, 36-, 32-, 27-, and 17-kDa (Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). We report the cloning and characterization of an additional V-ATPase subunit, the 54-kDa subunit, which is encoded by the VMA13 gene. VMA13 was isolated by complementation of the growth phenotypes associated with the vma13 mutation, which was originally described as cls11 (Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y. (1991) J. Biol. Chem. 266, 13971-13977). The nucleotide sequence of the VMA13 gene predicted a hydrophilic polypeptide with a calculated molecular mass of 54,415 daltons. The VMA13 54-kDa gene product resides on the vacuolar membrane and co-purified with the active V-ATPase complex. Characterization of a null vma13 mutant (delta vma13) revealed that the Vma13 polypeptide is essential for V-ATPase activity. However, the Vma13 polypeptide is not required for targeting of the other V-ATPase subunits (100-, 69-, 60-, 42-, 27-, or 17-kDa subunits) to the vacuolar membrane as shown by the association of these subunits with vacuolar membranes isolated from delta vma13 cells. The nature of the V-ATPase "complex" in delta vma13 mutant is, nevertheless, fundamentally different from the wild-type enzyme. This is evidenced by the fact that the inactive V-ATPase complex from delta vma13 cells is less stable than the wild-type enzyme. Taken together, these results indicate that VMA13 encodes the 54-kDa subunit of the V-ATPase and that this subunit is essential for activity, but not assembly, of the enzyme complex.

Amino Acid Sequence↗

PubMed1993

Structure of the heme-copper binuclear center of the cytochrome bo complex of Escherichia coli: EPR and Fourier transform infrared spectroscopic studies.

The cytochrome bo complex is a terminal quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump. To clarify the structural differences of the binuclear reaction center between the cytochrome bo complex and the mitochondrial cytochrome c oxidase, a combined study using EPR and Fourier transform infrared spectroscopies was carried out. The EPR spectrum of the highly purified cytochrome bo complex in the air-oxidized state showed a broad EPR signal (peak g* = 3.7) from an integer spin system. This confirms the existence of the spin-spin exchange-coupled binuclear site, in which the Feo3+ and CuB2+ centers were bridged by an unknown ligand (X). Binding of azide at the binuclear site as an ionic modulator weakened the strength of the spin-spin exchange coupling and thus caused a narrowing of the broad EPR signal. Binding of another modulator, formate, at the binuclear site caused the formation of EPR signals at g' = 12 and 2.7, which are very similar to those observed for cytochrome c oxidase. Cyanide replaced the bridging ligand (X) to form an Feo(3+)-C-N-CuB2+ structure in which strong spin-spin exchange coupling is expected, leading to a complete EPR-invisible state. Infrared evidence (a 2146 cm-1 C-N stretching band for the cyanide complex and a 2041 cm-1 azide antisymmetric stretching band for the azide complex) supported the theory that these ligands form bridging structures at the binuclear center, as previously observed for cytochrome c oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Azides↗

PubMed1993

VMA12 is essential for assembly of the vacuolar H(+)-ATPase subunits onto the vacuolar membrane in Saccharomyces cerevisiae.

vma12 mutants of the yeast Saccharomyces cerevisiae, which were originally identified as calcium-sensitive (cls) mutants that were also respiratory deficient (Pet-), have a defect in vacuolar membrane H(+)-ATPase activity (Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y. (1991) J. Biol. Chem. 266, 13971-13977). The VMA12 gene was cloned by complementation of the growth defects of vma12 mutants. The nucleotide sequence of the gene predicts a polypeptide of 215 amino acids (25.2 kDa) with two putative membrane-spanning domains. A null vma12 mutant, constructed by chromosomal deletion of the gene, is viable but has completely lost the vacuolar membrane H(+)-ATPase activity and exhibits the same growth defects as observed for the original vma12 mutants. Synthesis and targeting of the subunits of the H(+)-ATPase in the delta vma12 mutant cells were examined by Western blotting analyses of whole cell and vacuolar membrane protein extracts. None of the peripheral membrane subunits that we analyzed (the 69-, 60-, 42-, and 27-kDa subunits) was detected in the vacuolar membrane fractions, although the cellular levels of these polypeptides appeared to be normal. The 100- and 17-kDa integral membrane subunits of the enzyme were absent or present at a substantially reduced level in mutant vacuolar membrane fractions. Anti-Vma12p antibodies recognized a vacuolar protein with the expected molecular mass of 25 kDa. However, the Vma12 protein was not detected in the vacuolar membrane ATPase complex that had been solubilized with a zwitterionic detergent, ZW3-14, and purified by glycerol gradient centrifugation (Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). These results indicate that the VMA12 gene product is not a component of the active vacuolar ATPase complex and instead suggest that this protein is required during the process of assembly and/or targeting of the enzyme complex to the vacuolar membrane.

Amino Acid Sequence↗