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H Shoun

Publications and source records attributed to H Shoun.

At least 55 records · Page 3Linked to original sources
PubMed1994

Kinetics and thermodynamics of CO binding to cytochrome P450nor.

The CO-binding reaction of cytochrome P450nor isolated from denitrifying fungus, Fusarium oxysporum, has been studied by using a flash photolysis method in the millisecond time domain. We obtained the CO on- and off-rate constants in the bimolecular reaction, and determined the activation free energy, enthalpy, and entropy from the temperature dependence of these rate constants. To discuss the structural characteristics of P450nor, these parameters were compared with those of other cytochrome P450s, such as cytochrome P450cam from Pseudomonas putida and myoglobin. The on-rate constant (k(on)) for P450nor is larger than those of camphor-bound P450cam [P450cam(+)], suggesting that ligand entry to the heme pocket of P450nor is sterically less restricted than that of P450cam(+). In the P450norCO complex, the IR stretching band of the iron-bound CO is observed at 1942 cm-1, which is the same position as in P450cam(+)CO. This result suggests that the heme pocket immediate to the ligand-binding site is the same size in the two enzymes, in good agreement with the observation that the equilibrium constant (K = k(on)/k(off)) is identical in P450nor and P450cam(+). On the other hand, the entropy changes in the equilibrium and the off-activation processes are smaller in P450nor than in P450cam(+). This feature could reflect the lack of a bound substrate at the active site of P450nor. These structural characteristics of P450nor are discussed in relation to its unique catalytic property, rapid NO reduction to yield N2O.

Carbon Monoxide↗
PubMed1994

Fatty acid hydroxylase of the fungus Fusarium oxysporum is possibly a fused protein of cytochrome P-450 and its reductase.

Fatty acid subterminal (omega-1 approximately omega-3) hydroxylase of the fungus Fusarium oxysporum was solubilized from the microsomal fraction and partially purified. The hydroxylase activity was recovered into a single active fraction, and its spectral nature showed the presence of cytochrome P-450 (P-450). Fatty acid hydroxylase activity was markedly restored upon addition of FAD, FMN, and/or hemin to the eluted fraction. The fraction also exhibited other properties characteristic of both a hemeprotein and a flavin-containing reductase. These results are highly indicative that the fungal hydroxylase is a fused protein containing both P-450 and its reductase domains. In this aspect the fungal enzyme resembles bacterial P-450BM3, although it is membrane-bound unlike the bacterial counterpart.

Chromatography, Affinity↗
PubMed1994

Crystallization and preliminary X-ray diffraction studies of nitric oxide reductase cytochrome P450nor from Fusarium oxysporum.

Crystals of cytochrome P450nor (P450nor), nitric oxide reductase from Fusarium oxysporum, were obtained by means of the hanging-drop vapour diffusion technique in the presence of 1.88 M ammonium sulphate (pH 7.2). They belong to the monoclinic space group P2(1), with unit cell dimensions of a = 74.7 A, b = 86.7 A, c = 62.0 A and beta = 97 degrees. There are two molecules of P450nor in the asymmetric unit. The crystal diffracted to beyond 2.5 A resolution and is suitable for X-ray crystallographic study.

Crystallization↗
PubMed1994

Nitric oxide reductase cytochrome P-450 gene, CYP 55, of the fungus Fusarium oxysporum containing a potential binding-site for FNR, the transcription factor involved in the regulation of anaerobic growth of Escherichia coli.

A unique cytochrome P-450 (P-450) is involved in fungal denitrification, acting as a nitric oxide reductase. The phylogenetic classification of the P-450 (P-450nor) into the group of bacterial P-450s along with its involvement in the anaerobic process are of evolutional interest. The corresponding gene, CYP 55, of the fungus Fusarium oxysporum was isolated and sequenced. The P-450nor gene contained 7 introns that consisted of 49 to 55 bp. The presence of the introns suggested that horizontal transfer of the gene from bacteria to the fungus, if it has occurred, was an early event in evolution. Besides a TATA box, several inverted repeats were found in the 5'-upstream flanking region. One inverted repeat exhibited the same sequence as the binding site of FNR (fumarate and nitrate reduction), a DNA-binding, O2 (dioxygen)-sensor protein that positively regulates expressions of many hypoxic genes in Escherichia coli and other bacteria. The result suggested that the expression of P450nor is regulated in response to oxygen tension by an FNR-like system. Northern blot analyses showed that both nitrate and nitrite are the actual inducers of P-450nor and that its expression is predominantly regulated at the transcriptional level. These results raise the interesting possibility that the expression of the fungal denitrification system is regulated, as in the case of bacterial nitrate respiration, by a set of mechanisms, i.e., a combination of an FNR-like system and a system responding to nitrate/nitrite.

Amino Acid Sequence↗
PubMed1994

Levoglucosan dehydrogenase involved in the assimilation of levoglucosan in Arthrobacter sp. I-552.

A levoglucosan (1,6-anhydro-beta-D-glucopyranose)-using bacterium, isolated from soil, was identified. It was shown to belong to the genus Arthrobacter and tentatively named Arthrobacter sp. I-552. A novel enzyme catalyzed the dehydrogenation of levoglucosan to form 1,6-anhydro-beta-D-ribo-hexopyranos-3-ulose (3-keto levoglucosan), using NAD+ as an electron acceptor, i.e. NAD+: 1,6-anhydro-beta-D-glucopyranose oxidoreductase (trivial name: levoglucosan dehydrogenase). This enzyme was purified and characterized. A possible reaction scheme for the glucose formation was proposed. This pathway for levoglucosan use is distinct from those in yeast and fungi.

Amino Acid Sequence↗
PubMed1993

Expression of the fungal cytochrome P-450nor cDNA in Escherichia coli.

We succeeded in expressing the unique cytochrome P-450 (P-450), nitric oxide reductase (P-450nor), in Escherichia coli by utilizing P-450nor cDNA and the expression vector pKK233-2 or pUC19. The expression was confirmed by Western blot analysis and by detecting the unique nitric oxide reductase activity. The expressed protein was recovered in the soluble fraction of transformed cells, supporting that P-450nor is the first soluble P-450 of eukaryote. The results also provided conclusive evidence that the enzymatic reaction depends solely on P-450nor without support of any other electron transferring components.

Blotting, Western↗
PubMed1993

Cytochrome P-450 55A1 (P-450dNIR) acts as nitric oxide reductase employing NADH as the direct electron donor.

Cytochrome P-450dNIR (P-450dNIR), involved in the fungal denitrification by Fusarium oxysporum, was purified to homogeneity. The cytochrome P-450 (P-450) exhibited a potent nitric oxide (NO) reductase activity to form nitrous oxide (N2O) employing NADH but not NADPH as the sole effective electron donor. The apparent maximum turnover rate against NO was estimated as high as 31,500 min-1. The stoichiometry of the reaction between NO:NADH:N2O was 2:1:1. The reaction required neither an artificial electron-carrying mediator nor other proteinaceous components. An anaerobic incubation of ferric P-450dNIR.NO complex, but not of free ferric P-450dNIR, resulted in rapid reduction of the P-450, indicating that P-450dNIR.NO complex was reduced directly by NADH. Spectral changes during catalytic turnover indicated that decomposition of the ferrous P-450.NO complex might be rate-limiting. The reaction was not inhibited by carbon monoxide at all, suggesting that the free ferrous P-450 is not formed during turnover. On the basis of these results a possible reaction mechanism was considered. The present results demonstrated not only the unique reaction catalyzed by P-450, but also the first P-450-dependent reaction where the electron transport from NAD(P)H to P-450 is not supported by other components. Now that the physiological function of P-450 55A1 (P-450dNIR) has been found, we propose to alter the trivial name to P-450nor.

Chromatography, DEAE-Cellulose↗
PubMed1992

Denitrification by fungi.

Many fungi in the centre of the group of Fusarium and its teleomorphs were shown to be capable of reducing nitrite anaerobically to form nitric oxide (NO), nitrous oxide (N2O), and/or dinitrogen (N2). Several strains could reduce nitrate as well. Nitrous oxide was the major product of the reduction of nitrate or nitrite. Several fungi could also form N2. When [15]nitrite was used as substrate for the N2-forming denitrification, 15N2O, 15NO, and 14N15N were obtained as the products. These results demonstrated that, unexpectedly, many fungi have denitrifying abilities. It was also shown that the fungal system contains a unique reaction, formation of a hybrid dinitrogen.

Anaerobiosis↗
PubMed1992

Analysis of the transcriptional activity of the hut promoter in Bacillus subtilis and identification of a cis-acting regulatory region associated with catabolite repression downstream from the site of transcription.

Levels of transcripts initiated at a hut promoter in Bacillus subtilis were analysed. The addition of histidine to the culture medium increased the level of the transcript sixfold. In the presence of histidine and glucose together, the level of the transcript was reduced to the level in the absence of induction. Furthermore, addition of a mixture of 16 amino acids to cultures of induced cells and of catabolite-repressed cells decreased levels of the transcript 16-fold and 2.6-fold, respectively. Thus, it appears that at least three regulatory mechanisms associated with induction, catabolite repression, and amino acid repression, control the transcriptional activity of the hut promoter. Expression of the hut promoter-lacZ fusions that contained various regions of the hutP gene and deletion analysis of the hutP region revealed a cis-acting sequence associated with catabolite repression that was located between positions +204 and +231 or around position +203.

Amino Acids↗
PubMed1991

Denitrification by the fungus Fusarium oxysporum and involvement of cytochrome P-450 in the respiratory nitrite reduction.

From conditions for production in Fusarium oxysporum of the unique nitrate/nitrite-inducible cytochrome P-450, tentatively called P-450dNIR, it was expected that the fungus is capable of metabolizing nitrate dissimilatively. Here we report that F. oxysporum exhibits a distinct denitrifying ability which results in the anaerobic evolution of nitrous oxide (N2O) from nitrate or nitrite. Comparison of the cell growth during denitrification indicated that the dissimilatory reduction of nitrate to nitrite is an energetically favorable process in F. oxysporum; however, further reduction of nitrite to N2O might be energy-exhausting and may function as a detoxification mechanism. A potent nitrite reductase activity to form N2O could be reconstituted by combination of the cell-free extract prepared from the denitrifying cells and an NADH-phenadinemethosulfate-dependent reducing system. The activity was strongly inhibited by carbon monoxide, cyanide, oxygen (O2), and the antibody against P-450dNIR. The results, along with those concerning inducing conditions of P-450dNIR, were highly indicative that the cytochrome is involved in the denitrifying nitrite reduction. This work has thus presented not only the first demonstration that a eukaryote exhibits a marked denitrifying ability, but also the first instance of a cytochrome P-450 that is involved in a reducing reaction with a distinct physiological significance against a hydrophilic, inorganic substrate.

Cytochrome P-450 Enzyme System↗
PubMed1991

Nucleotide sequence of the unique nitrate/nitrite-inducible cytochrome P-450 cDNA from Fusarium oxysporum.

A cDNA clone for the nitrate/nitrite-inducible cytochrome P-450 (P-450) of the fungus Fusarium oxysporum (tentatively termed P-450dNIR) was isolated by an immunoscreening method. Sequence determination revealed a polypeptide of 403 amimo acid residues (Mr = 44,371), which was shown to contain the full-length sequence of the fungal P-450. The amino terminus region of the predicted sequence contained neither the signal-like, hydrophobic domain that is commonly observed in microsomal P-450s nor the tagging prosequence that is essential for localization of mitochondrial P-450s. Further, the sequence exhibited higher homologies against those of soluble bacterial P-450s, in particular P-450s of Streptomyces, rather than those of eukaryotic P-450s including yeast and fungal P-450s. These results are highly indicative that P-450dNIR is the first soluble P-450 derived from eukaryotic organisms. The unique features might be related to the novel function of P-450dNIR, which is involved in a dissimilatory reduction of nitrite by the fungus. P-450dNIR was classified into a new family, P-450LV, and the corresponding gene of the fungus was named CYP55.

Amino Acid Sequence↗
PubMed1989

Soluble, nitrate/nitrite-inducible cytochrome P-450 of the fungus, Fusarium oxysporum.

Both soluble and microsomal fractions of Fusarium oxysporum contain cytochrome P-450(P-450). We report here that the P-450 in the soluble fraction was induced only when nitrate or nitrite was added to the growth medium, whereas the microsomal P-450 was synthesized regardless of the medium compositions. The reduced-CO complex of the soluble P-450 exhibited an absorption spectrum that is different from that of the microsomal counterpart. These results indicate that the soluble P-450 is distinct from the microsomal species and suggest a novel function for the former P-450.

Carbon Monoxide↗
PubMed1986

Cloning and sequencing of a gene for Mucor rennin, an aspartate protease from Mucor pusillus.

The aspartate protease of Mucor pusillus (Mucor pusillus rennin; MPR) is a milk-clotting enzyme used in the cheese industry. The partial amino acid sequence of MPR was determined and oligonucleotide probes were synthesized for cloning of the MPR gene. A clone giving positive hybridization with the probes was selected from the cosmid library. Sequencing of the cloned DNA revealed an open reading frame of 1281 bp without introns which encodes 361 amino acids for the expected MPR with an NH2-terminal extension of 66 amino acids. MPR seems to be synthesized as a prepro enzyme.

Amino Acid Sequence↗
PubMed1985

Subterminal hydroxylation of fatty acids by a cytochrome P-450-dependent enzyme system from a fungus, Fusarium oxysporum.

The cell-free extract of a cytochrome P-450-producing fungus, Fusarium oxysporum, was found to catalyze the hydroxylation of fatty acids. Three product isomers were formed from a single fatty acid. The products from lauric acid were identified by mass-spectrometry as 9-, 10-, and 11-hydroxydodecanoic acids, and those from palmitic acid as 13-, 14-, and 15-hydroxyhexadecanoic acids. The ratio of the isomers formed was 50 : 36 : 14 in the case of laurate hydroxylation, and 37 : 47 : 16 in the case of palmitate. The reaction was dependent on both NADPH (or NADH) and molecular oxygen,and was strongly inhibited by carbon monoxide, menadione, or the antibody to purified Fusarium P-450. Further, lauric acid induced a type I spectral change in purified Fusarium P-450. Further, lauric acid induced a type I spectral change in purified Fusarium P-450 with an apparent Kd of 0.3 mM. The hydroxylase activity together with cytochrome P-450 could be detected in both the soluble and microsome fractions, and the activity was almost proportional to the amount of cytochrome P-450 reducible with NADPH. It can be concluded from these results that Fusarium P-450 reducible with NADPH. It can be concluded from these results that Fusarium P-450 is involved in the (omega-1)-, (omega-2)-, and (omega-3)-hydroxylation of fatty acids catalyzed by the cell-free extract of the fungus.

Antibodies↗
PubMed1983

Inhibition of p-hydroxybenzoate hydroxylase by anions: possible existence of two anion-binding sites in the site for reduced nicotinamide adenine dinucleotide phosphate.

Certain anions were found to inhibit p-hydroxybenzoate hydroxylase from Pseudomonas desmolytica. The inhibition was of competitive or mixed type with respect to NADPH (apparent Ki = 4-30 mM). Among the anions, monovalent anions such as halogen ions and azide inhibited ionization of the phenolic hydroxyl group of the substrate (p-hydroxybenzoate) on binding with the enzyme . substrate complex of p-hydroxybenzoate hydroxylase, without dissociating the substrate from the enzyme. On the other hand, multivalent anions (anions of polybasic acids), such as inorganic phosphate, borate, and sulfate, did not inhibit the ionization. Halogen ions induced remarkable spectral changes in the FAD moiety of the enzyme on binding, while the change due to inorganic phosphate was only slight. Chloride inhibited the binding of NADH with the enzyme as well as that of NADPH, whereas borate inhibited the binding of only NADPH. These results indicate that the monovalent and multivalent anions probably bind to the sites in the enzyme which interact, respectively, with the pyrophosphate and 2'-phosphate moieties of NADPH. The results provide strong support for the catalytic mechanism in which the phenolate anion of p-hydroxybenzoate participates in the process of substrate hydroxylation by C (4a) peroxyflavin. The results also suggest that repeated ionization/neutralization of the phenolic hydroxyl group of the substrate may occur during one cycle of the catalytic turnover.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1983

Purification and properties of a cytochrome P-450 of a fungus, Fusarium oxysporum.

A cytochrome P-450 was isolated from a fungus, Fusarium oxysporum, which grew on a medium containing soybean oil as a sole carbon source. It was found as a heme protein that possess lipoxygenase activity, and seemed to exist in the soluble fraction of cell-free extracts. The cytochrome revealed multiplicity and could be separated into a least 3 fractions (A, B, and C). Two of them, termed Fusarium P-450A and -B, were highly purified. The complex of the ferrous Fusarium P-450 with carbon monoxide showed a Soret peak at 447 nm. The properties of the cytochromes (P-450A and -b) were closely similar to each other, the only detectable difference being in the pI (isoelectric point) value (5.2 and 5.0, respectively). The pI and molecular weight (48,000) values together with amino acid composition of Fusarium P-450 were similar to those of other cytochromes P-450 from various sources. Some other spectral properties as well as interactions with various ligands were also studied. Peroxidase or chloroperoxidase activity was not detected with Fusarium P-450.

Amino Acids↗
PubMed1982

A histidine residue in p-hydroxybenzoate hydroxylase essential for binding of reduced nicotinamide adenine dinucleotide phosphate.

Chemical modification with diethylpyrocarbonate (ethoxyformic anhydride) was examined to demonstrate the existence of an essential histidine residue at the NADPH-binding site of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas desmolytica. Among some ligands, NADPH was noticeable in protecting the enzyme from the modification-caused inactivation. Although several amino acid residues were modified during the inactivation process, inhibition of the enzyme could be correlated with modification of a single histidine residue which was masked by addition of NADPH. The pK of the essential histidine residue was estimated to be 6.5-6.7. The Kd (Km) for NADPH of the inactivated enzyme was shown to have been increased greatly, although the Kd for substrate (p-hydroxybenzoate) was not changed.

4-Hydroxybenzoate-3-Monooxygenase↗