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O Adachi

Publications and source records attributed to O Adachi.

At least 55 records · Page 3Linked to original sources
PubMed1996

Comparison of the structural features of ubiquinone reduction sites between glucose dehydrogenase in Escherichia coli and bovine heart mitochondrial complex I.

To characterize the structural features of the ubiquinone reduction site of glucose dehydrogenase (GlcDH) in Escherichia coli, we performed structure/activity studies of a systematic set of synthetic ubiquinone analogues and specific inhibitors (synthetic capsaicins) of this site. Considering the proposed similarity of the quinone binding domain motif between GlcDH and one subunit of mitochondrial complex I [Friedrich, T., Strohdeicher, M., Hofhaus, G., Preis, D., Sahm, H. & Weiss, H. (1990) FEBS Lett. 265, 37-40], we compared the structure/activity profiles of the substrates and inhibitors for GlcDH with those for bovine heart mitochondrial complex i. With respect to GlcDH, replacement of one or both methoxy groups in the 2 and 3 positions of benzoquinone ring by ethoxy group(s) resulted in a drastic decrease in the electron accepting activity. The presence of a 5-methyl group and the conformational property of the 6-alkyl side chain did not significantly contribute to the activity. These results suggested that only half of the benzoquinone ring (the moiety corresponding to the 2 and 3 positions) is recognized by the quinone reduction site in a strict sense. In contrast, quinone analogues with structural modifications at all positions in the benzoquinone ring retained the activity with mitochondrial complex I. This finding indicated that the catalytic site of complex I is spacious enough to accommodate a variety of structurally different quinone derivatives. The correlation of the inhibitory potencies of a series of synthetic capsaicins between the two enzymes was very poor. These findings indicated that the binding environment of ubiquinone in GlcDH is very specific and differs from that in mitochondrial complex I.

Animals↗
PubMed1996

Two distinct quinoprotein amine oxidases are induced by n-butylamine in the mycelia of Aspergillus niger AKU 3302. Purification, characterization, cDNA cloning and sequencing.

Two distinct quinoprotein amine oxidases were found in Aspergillus niger mycelia grown on n-butylamine medium and purified using chromatographic techniques. The respective enzymes were termed AO-I, which had already been isolated, and AO-II, a new enzyme found in this study. HPLC indicated that their molecular masses are 150 kDa and 80 kDa, respectively. On SDS/PAGE, the enzymes gave a similar but distinct mobility, which corresponds to 75 kDa for the subunit dimeric AO-I and 80 kDa for monomeric AO-II. The absorption spectra of both enzymes were different from each other; the absorption maxima in the visible region were at 490 nm for AO-I and 420 nm for AO-II. The enzymes showed positive quinone staining, comparable substrate specificity, and sensitivity to inhibitors typical for copper/topa quinone-containing amine oxidases, but they had different copper contents and also differed in their N-terminal sequences. Their peptide maps showed almost identical patterns, with the exception of two additional bands for AO-II. Among the peptides obtained from digestion of AO-II, peptides with sequences corresponding to the N-terminal part of AO-I were detected. Polyclonal antibodies raised against AO-I and AO-II recognized both enzymes, but with different specificities. Using precipitation with AO-I, the antibody prepared against AO-II was purified and was shown to be specific only for AO-II. The cDNA of AO-I was cloned and sequenced. A highly conserved tetrapeptide sequence, Asn-Tyr-Glu-Tyr, was identified in which the first tyrosine residue (Tyr404) that could be converted to topa quinone was present in the 670-residue deduced amino acid sequence. Northern blot analysis indicated that AO-I was highly expressed in A. niger grown on n-butylamine as a single nitrogen source. Genomic Southern blot analysis confirmed that both enzymes are likely to be encoded by the same gene.

Amine Oxidase (Copper-Containing)↗
PubMed1996

Function of multiple heme c moieties in intramolecular electron transport and ubiquinone reduction in the quinohemoprotein alcohol dehydrogenase-cytochrome c complex of Gluconobacter suboxydans.

Alcohol dehydrogenase (ADH) of acetic acid bacteria functions as the primary dehydrogenase of the ethanol oxidase respiratory chain, where it donates electrons to ubiquinone. ADH is a membrane-bound quinohemoprotein-cytochrome c complex which consists of subunits I (78 kDa), II (48 kDa), and III (14 kDa) and contains several hemes c as well as pyrroloquinoline quinone as prosthetic groups. To understand the role of the heme c moieties in the intramolecular electron transport and the ubiquinone reduction, the ADH complex of Gluconobacter suboxydans was separated into a subunit I/III complex and subunit II, then reconstituted into the complex. The subunit I/III complex, probably subunit I, contained 1 mol each of pyrroloquinoline quinone and heme c and exhibited significant ferricyanide reductase, but no Q1 reductase activities. Subunit II was a triheme cytochrome c and had no enzyme activity, but it enabled the subunit I/III complex to reproduce the Q1 and ferricyanide reductase activities. Hybrid ADH consisting of the subunit I/III complex of G. suboxydans ADH and subunit II of Acetobacter aceti ADH was constructed and it had showed a significant Q1 reductase activity, indicating that subunit II has a ubiquinone-binding site. Inactive ADH from G. suboxydans exhibiting only 10% of the Q1 and ferricyanide reductase activities of the active enzyme has been isolated separately from active ADH (Matsushita, K., Yakushi, T., Takaki, Y., Toyama, H., and Adachi, O (1995) J. Bacteriol. 177, 6552-6559). Using these active and inactive ADHs and also isolated subunit I/III complex, we performed kinetic studies which suggested that ADH contains four ferricyanide-reacting sites, one of which was detected in subunit I and the others in subunit II. One of the three ferricyanide-reacting sites in subunit II was defective in inactive ADH. The ferricyanide-reacting site remained inactive even after alkali treatment of inactive ADH and also after reconstituting the ADH complex from the subunits, in contrast to the restoration of Q1 reductase activity and the other ferricyanide reductase activities. Thus, the data suggested that the heme c in subunit I and two of the three heme c moieties in subunit II are involved in the intramolecular electron transport of ADH into ubiquinone, where one of the two heme c sites may work at, or close to, the ubiquinone-reacting site and another between that and the heme c site in subunit I. The remaining heme c moiety in subunit II may have a function other than the electron transfer from ethanol to ubiquinone in ADH.

Acetobacter↗
PubMed1995

Generation mechanism and purification of an inactive form convertible in vivo to the active form of quinoprotein alcohol dehydrogenase in Gluconobacter suboxydans.

Alcohol dehydrogenase (ADH) of acetic acid bacteria is a membrane-bound quinohemoprotein-cytochrome c complex involved in vinegar production. In Gluconobacter suboxydans grown under acidic growth conditions, it was found that ADH content in the membranes was largely increased but the activity was not much changed, suggesting that such a condition produces an inactive form of ADH (inactive ADH). A similar phenomenon could be also observed in Acetobacter aceti, another genus of acetic acid bacteria. Furthermore, aeration conditions were also shown to affect ADH production; the ADH level was increased and was present as an active form under low-aeration conditions, while the ADH level was decreased and was present mainly as an inactive form under high-aeration conditions. Inactive ADH was solubilized from the membranes of G. suboxydans grown in acidic and high-aeration conditions and was purified separately from the normal, active form of ADH (active ADH). In spite of having 10 times less enzyme activity than active ADH, inactive ADH could not be distinguished from active ADH with respect to their subunit compositions, molecular sizes, and prosthetic groups. Inactive ADH, however, had a relatively loose conformation with a partially oxidized state, while active ADH had a tight conformation with a completely reduced state, suggesting that inactive ADH may lack a right subunit's interaction and that one of the heme c components may be inactivated. Reactivation from such an inactive ADH occurred either by shifting of the pH of the culture medium up during the cultivation or by incubation of the resting cells at the neutral pH region in the presence of an energy source such as D-sorbitol. Such an activation of ADH was repressed by the addition of a proton uncoupler and could not occur in the spheroplasts. Thus, the results suggest that inactive ADH could be generated abundantly under acidic growth conditions and converted to the active form at a neutral culture pH. The data also suggest that some periplasmic component may be involved in the conversion of inactive ADH into the active form by consuming some forms of energy.

Acetobacteraceae↗
PubMed1995

Three distinct quinoprotein alcohol dehydrogenases are expressed when Pseudomonas putida is grown on different alcohols.

A bacterial strain that can utilize several kinds of alcohols as its sole carbon and energy sources was isolated from soil and tentatively identified as Pseudomonas putida HK5. Three distinct dye-linked alcohol dehydrogenases (ADHs), each of which contained the prosthetic group pyrroloquinoline quinone (PQQ), were formed in the soluble fractions of this strain grown on different alcohols. ADH I was formed most abundantly in the cells grown on ethanol and was similar to the quinoprotein ADH reported for P. putida (H. Görisch and M. Rupp, Antonie Leeuwenhoek 56:35-45, 1989) except for its isoelectric point. The other two ADHs, ADH IIB and ADH IIG, were formed separately in the cells grown on 1-butanol and 1,2-propanediol, respectively. Both of these enzymes contained heme c in addition to PQQ and functioned as quinohemoprotein dehydrogenases. Potassium ferricyanide was an available electron acceptor for ADHs IIB and IIG but not for ADH I. The molecular weights were estimated to be 69,000 for ADH IIB and 72,000 for ADH IIG, and both enzymes were shown to be monomers. Antibodies raised against each of the purified ADHs could distinguish the ADHs from one another. Immunoblot analysis showed that ADH I was detected in cells grown on each alcohol tested, but ethanol was the most effective inducer. ADH IIB was formed in the cells grown on alcohols of medium chain length and also on 1,3-butanediol. Induction of ADH IIG was restricted to 1,2-propanediol or glycerol, of which the former alcohol was more effective. These results from immunoblot analysis correlated well with the substrate specificities of the respective enzymes. Thus, three distinct quinoprotein ADHs were shown to be synthesized by a single bacterium under different growth conditions.

1-Butanol↗
PubMed1995

Inhibition of copper/quinoprotein amine oxidases from Aspergillus niger by benzophenanthridine alkaloids.

Inhibition of copper/quinoprotein amine oxidases (EC 1.4.3.6), AO-I (dimer 2 x 75 kDa) and AO-II (monomer 80 kDa), from the fungus Aspergillus niger by benzophenanthridine alkaloids sanguinarine, chelerythrine, and fagaronine were studied. For both amine oxidases the alkaloids showed reversible noncompetitive inhibition of n-hexylamine oxidation with Ki 0.6, 0.9 and 2.8 mM for sanguinarine, chelerythrine, and fagaronine, respectively. The values of the inhibition constants corresponded to pKR+ values for the iminium ion/pseudobase equilibrium of the alkaloids. Since thio-compounds protected the enzymes against this inhibition, the inhibition effect was ascribed to the interaction with a sulfhydryl group essential for the enzymatic activity.

Alkaloids↗
PubMed1995

Half-site reactivity with p-nitrophenylhydrazine and subunit separation of the dimeric copper-containing amine oxidase from Aspergillus niger.

Structural properties of dimeric (2 x 75 kDa) copper-containing amine oxidase (EC 1.4.3.6) from Aspergillus niger were studied. The enzyme treated with SDS was dissociated into subunits which showed different mobility on polyacrylamide gel without SDS. The separated subunits had no activity but a quinone moiety was detected in both by a redox-cyclic quinone staining. After titration of the enzyme with p-nitrophenylhydrazine, which showed half-site reactivity (1 mole per dimer), and SDS treatment both p-nitro-phenylhydrazone and a remaining quinone moiety were detected in each subunit. It is suggested that the half-site reactivity with phenylhydrazine is caused by conformational changes after binding of the inhibitor to any one of the active sites leading to inaccessibility of the second active site for the inhibitor. The difference in electrophoretic mobility of the separated subunits originates probably from their structural difference likely to occur outside the active site, even if the amino acid sequences of the subunits appear to be identical.

Amine Oxidase (Copper-Containing)↗
PubMed1994

Structure-function studies on the ubiquinol oxidation site of the cytochrome bo complex from Escherichia coli using p-benzoquinones and substituted phenols.

To characterize the structural features of the quinol oxidation site (the QL site) of the cytochrome bo complex, a heme-copper respiratory oxidase in Escherichia coli, we carried out structure-inhibitory potency analyses using 7 p-benzoquinones and 33 substituted phenols. Their effects on its ubiquinol-1 oxidase activity were compared with those on the cytochrome bd complex in E. coli and on cytochromes o and alpha 1 in Acetobacter aceti. They showed similar structural properties of the QL site, although cytochrome o was more sensitive to 4-cyanophenols, suggesting a specific interaction of the hydrogen bond-accepting cyano group with the binding pocket. Replacing one of the methyl groups of 2,6-dimethyl-p-benzoquinone, which is the most potent competitive inhibitor, with an ethyl group markedly decreased the inhibitory activity, indicating that the QL site specifically recognizes one C = O group with two methyl groups as the ortho-substituents. In substituted phenols, ortho-chlorine substituents were the most effective in recognition, and the electron-withdrawing ability of the para-substituent determined an inhibitory potency, probably by stabilizing an anionic form. Based on these observations, we postulate that the QL site of the cytochrome bo complex asymmetrically recognizes exogenous ligands and that this property accounts for the sequential electron transfer from ubiquinols to the low-spin heme.

Benzoquinones↗
PubMed1994

Active-site covalent modifications of quinoprotein amine oxidases from Aspergillus niger. Evidence for binding of the mechanism-based inhibitor, 1,4-diamino-2-butyne, to residue Lys356 involved in the catalytic cycle.

Interactions of two distinct quinoprotein amine oxidases from Aspergillus niger, AO-I and AO-II, with active-site covalent modifiers have been investigated. Both enzymes are inhibited similarly by phenylhydrazine or p-nitrophenylhydrazine, forming an orange Schiff base with a carbonyl group of topaquinone cofactor. Modification of histidyl and tyrosyl residues by diethylpyrocarbonate and sulfhydryl groups by 5,5'-dithio-bis-(2-nitrobenzoic acid) and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole have been described. A substrate analog, 1,4-diamino-2-butyne, was found to function as a mechanism-based inhibitor. It shows both substrate saturation kinetics and time-dependent irreversible inhibition caused by formation of pyrrole bound to the active site. The pyrrole formation was confirmed spectrophotometrically by reaction with Ehrlich's reagent at 525 nm. Inhibition by 1,4-diamino-2-butyne produces a new maximum in the absorption spectra of AO-I and AO-II at 310 nm and 306 nm, respectively. Inactivated AO-I was digested by proteases; labeled peptides were purified by C18 HPLC and sequenced by Edman degradation. Data reveal the evidence that 1,4-diamino-2-butyne reacts with the epsilon-amino group of the Lys356 residue in the sequence Lys-Met-Pro-Asn-Ala of Aspergillus niger amine oxidase AO-I.

Amine Oxidase (Copper-Containing)↗
PubMed1993

Topological analysis of quinoprotein glucose dehydrogenase in Escherichia coli and its ubiquinone-binding site.

Topological structure of quinoprotein glucose dehydrogenase in the inner membrane of Escherichia coli was determined by constructing protein fusions with alkaline phosphatase or beta-galactosidase. Analysis of the fusions revealed that the dehydrogenase possesses five membrane-spanning segments, and the N-terminal and C-terminal portions resided at the cytoplasmic and periplasmic side of the membrane, respectively. These results agreed with the hydropathy profile based on its primary structure. The topological structure suggests that the predicted binding site of the prosthetic group pyrroloquinoline quinone is located at the periplasmic side and that the amino acid residues corresponding to those that were presumed to interact with ubiquinone in one subunit of mitochondrial NADH dehydrogenase also occur at the periplasmic side. When the purified glucose dehydrogenase and cytochrome o ubiquinol oxidase were reconstituted together with ubiquinone into liposomes, a membrane potential could be generated by the electron transfer at the site of the ubiquinol oxidase but not of the dehydrogenase. These results suggest that glucose dehydrogenase has a ubiquinone reacting site close to the periplasmic side of the membrane, and thus its electron transfer to ubiquinone appears to be incapable of forming a proton electrochemical gradient across the inner membrane of E. coli.

Alkaline Phosphatase↗
PubMed1993

A novel quinoprotein methanol dehydrogenase containing an additional 32-kilodalton peptide purified from Acetobacter methanolicus: identification of the peptide as a MoxJ product.

Acetobacter methanolicus is a unique acetic acid bacterium which has a methanol oxidase respiratory chain in addition to an ethanol oxidase respiratory chain. In this study, two different forms of methanol dehydrogenase (type I and II MDHs) were purified from A. methanolicus grown on methanol. Type I MDH was more basic (pI of 8.0) and smaller (M(r) of 148K) than type II MDH (pI of 6.7 and M(r) of 177K). Type I MDH consisted of alpha and beta subunits of 62 and 10 kDa, which has the same alpha 2 beta 2 conformation as the enzymes purified so far. The type II MDH contained an additional peptide of 32 kDa, of which a single copy was estimated to bind to the alpha 2 beta 2-MDH judging from the whole molecular weight and the stoichiometry of each subunit determined in sodium dodecyl sulfate-high-performance gel filtration chromatography. Compared with type I MDH, type II MDH exhibited a lower enzyme activity, but the electron-transfer activity to cytochrome cL was much more resistant to the inhibition with NaCl or EDTA. The possibility could be excluded that type II MDH is an artificial complex of type I MDH with a 32-kDa peptide, since it was inducible with methanol and could be detected in the periplasm, as well as the other subunits. Furthermore, the N-terminal amino acid sequence of the 32-kDa peptide showed a high homology to that of the moxJ product deduced from the DNA sequence of Paracoccus denitrificans or Methylobacterium extorquens AM1.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetobacter↗
PubMed1992

Homology in the structure and the prosthetic groups between two different terminal ubiquinol oxidases, cytochrome a1 and cytochrome o, of Acetobacter aceti.

Acetobacter aceti produces two different terminal oxidases dependent on the culture conditions, shaking and static cultures. Cells grown on shaking culture contain cytochrome a1, while cytochrome o is present in cells grown on static culture. Cytochrome a1 and cytochrome o of A. aceti were compared especially with respect to the protein structure and the prosthetic groups. Cytochrome a1 exhibited lower CN sensitivity and higher affinity for O2 than cytochrome o. Both terminal oxidases consisted of four nonidentical polypeptides of which the molecular sizes were identical between both enzymes. Cytochrome a1 cross-reacted with an antibody raised against cytochrome o at the same level as cytochrome o did, and an antibody elicited against cytochrome a1 cross-reacted with both cytochrome o and cytochrome a1 at the same intensity, which indicates that both oxidases are indistinguishable immunochemically. Furthermore, almost the same peptide mapping pattern with chymotrypsin was observed in subunit I and in subunit II between both terminal oxidases, and the amino-terminal sequences in the subunit II of both oxidases were identical at least in their 10 amino acids. As for the prosthetic groups, both oxidases were shown to contain two heme-irons and one copper atom. Further, high performance liquid chromatography analysis of the heme moieties extracted from both the purified enzymes indicated that cytochrome a1 contains hemes b and a at a ratio of 1 to 1, whereas cytochrome o contains the same amounts of hemes b and o. Thus, data indicate that cytochrome a1 and cytochrome o of A. aceti are cytochrome ba and cytochrome bo ubiquinol oxidases, respectively, and that both oxidases have a closely similar protein structure and prosthetic groups, in which only heme a in the heme/copper binuclear center of cytochrome a1 is replaced by heme o in that of cytochrome o.

Acetobacter↗
PubMed1992

Methanol and ethanol oxidase respiratory chains of the methylotrophic acetic acid bacterium, Acetobacter methanolicus.

Acetobacter methanolicus is a unique acetic acid bacterium which has a methanol oxidase respiratory chain, as seen in methylotrophs, in addition to its ethanol oxidase respiratory chain. In this study, the relationship between methanol and ethanol oxidase respiratory chains was investigated. The organism is able to grow by oxidizing several carbon sources, including methanol, glycerol, and glucose. Cells grown on methanol exhibited a high methanol-oxidizing activity and contained large amounts of methanol dehydrogenase and soluble cytochromes c. Cells grown on glycerol showed higher oxygen uptake rate and dehydrogenase activity with ethanol but little methanol-oxidizing activity. Furthermore, two different terminal oxidases, cytochrome c and ubiquinol oxidases, have been shown to be involved in the respiratory chain; cytochrome c oxidase predominates in cells grown on methanol while ubiquinol oxidase predominates in cells grown on glycerol. Both terminal oxidases could be solubilized from the membranes and separated from each other. The cytochrome c oxidase and the ubiquinol oxidase have been shown to be a cytochrome co and a cytochrome bo, respectively. Methanol-oxidizing activity was diminished by several treatments that disrupt the integrity of the cells. The activity of the intact cells was inhibited with NaCl and/or EDTA, which disturbed the interaction between methanol dehydrogenase and cytochrome c. Ethanol-oxidizing activity in the membranes was inhibited with 2-heptyl-4-hydroxyquinoline N-oxide, which inhibited ubiquinol oxidase but not cytochrome c oxidase. Alcohol dehydrogenase has been purified from the membranes of glycerol-grown cells and shown to reduce ubiquinone-10 as well as a short side-chain homologue in detergent solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetobacter↗
PubMed1992

Change of the terminal oxidase from cytochrome a1 in shaking cultures to cytochrome o in static cultures of Acetobacter aceti.

Acetobacter aceti has an ability to grow under two different culture conditions, on shaking submerged cultures and on static pellicle-forming cultures. The respiratory chains of A. aceti grown on shaking and static cultures were compared, especially with respect to the terminal oxidase. Little difference was detected in several oxidase activities and in cytochrome b and c contents between the respiratory chains of both types of cells. Furthermore, the results obtained here suggested that the respiratory chains consist of primary dehydrogenases, ubiquinone, and terminal ubiquinol oxidase, regardless of the culture conditions. There was a remarkable difference, however, in the terminal oxidase, which is cytochrome a1 in cells in shaking culture but cytochrome o in cells grown statically. Change of the culture condition from shaking to static caused a change in the terminal oxidase from cytochrome a1 to cytochrome o, which is concomitant with an increase of pellicle on the surface of the static culture. In contrast, reappearance of cytochrome a1 in A. aceti was attained only after serial successive shaking cultures of an original static culture; cytochrome a1 predominated after the culture was repeated five times. In the culture of A. aceti, two different types of cells were observed; one forms a rough-surfaced colony, and the other forms a smooth-surfaced colony. Cells of the former type predominated in the static culture, while the cells of the latter type predominated in the shaking culture. Thus, data suggest that a change of the culture conditions, from static to shaking or vice versa, results in a change of the cell type, which may be related to the change in the terminal oxidase from cytochrome a1 to cytochrome o in A. aceti.

Acetobacter↗
PubMed1992

Characterization of membrane-bound spermidine dehydrogenase of Citrobacter freundii.

Spermidine dehydrogenase found in the membrane fraction of Citrobacter freundii IFO 12681 was solubilized with Triton X-100 and further purified to homogeneity. The properties of the membrane enzyme were almost identical to those obtained from the soluble fraction of the organism with respect to molecular and catalytic properties. Thus, binding properties of the enzyme to the bacterial membrane were checked. The ratio of enzyme activity found in the soluble fraction to the membrane fraction was dependent on salt concentration during cell disruption. A hydrophobic interaction was largely involved in anchoring the enzyme to the membrane fraction. Purified spermidine dehydrogenase from the soluble fraction was readily adsorbed into the membrane fraction in the presence of salt. Spermidine dehydrogenase appeared to be a membrane-bound enzyme localized in the cytoplasmic membranes in a manner that makes a partial release of the enzyme possible during mechanical cell disruption. When spermidine oxidation was done with the resting cells of C. freundii, a stoichiometric formation of two reaction products, 1,3-diaminopropane and gamma-aminobutyraldeyde, was observed without any lag time. These facts indicate that the enzyme is localized on the outer surface of the cytoplasmic membranes or in the periplasmic space of the organism.

Amino Acids↗