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1.
Soluble, nitrate/nitrite-inducible cytochrome P-450 of the fungus, Fusarium oxysporum 总被引:2,自引:0,他引:2
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. 相似文献
2.
Nitric oxide reduction, the last step in denitrification by Fusarium oxysporum, is obligatorily mediated by cytochrome P450nor 总被引:2,自引:0,他引:2
The involvement of cytochrome P450nor (P450nor) is the most striking feature of the fungal denitrifying system, and has never
been shown in bacterial systems. To establish the physiological significance of the P450nor, we constructed and investigated
mutants of Fusarium oxysporum that lacked the gene for P450nor. We mutated the gene by targeted integration of a disrupted gene into the chromosome of
F. oxysporum. The mutants were shown to contain neither P450nor protein nor nitric oxide (NO) reductase (Nor) activity, implying that
they are indeed deficient in P450nor. These mutants had apparently lost the denitrifying activity and failed to evolve nitrous
oxide (N2O) upon incubation under oxygen-limiting conditions in the presence of nitrate. Their mycelia exhibited normal levels of dissimilatory
nitrite reductase (Nir) activity and were able to evolve NO under these conditions. The promoter region of the P450nor gene
was fused to lacZ and introduced into the wild-type strain of F. oxysporum. The transformed strain produced β-galactosidase under denitrifying conditions as efficiently as the wild type does P450nor.
These results represent unequivocal genetic evidence that P450nor is essential for the reduction of NO to N2O, the last step in denitrification by F. oxysporum.
Received: 28 June 1999 / Accepted: 22 December 1999 相似文献
3.
Nucleotide sequence of the unique nitrate/nitrite-inducible cytochrome P-450 cDNA from Fusarium oxysporum 总被引:5,自引:0,他引:5
H Kizawa D Tomura M Oda A Fukamizu T Hoshino O Gotoh T Yasui H Shoun 《The Journal of biological chemistry》1991,266(16):10632-10637
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. 相似文献
4.
Denitrification by the fungus Fusarium oxysporum and involvement of cytochrome P-450 in the respiratory nitrite reduction 总被引:7,自引:0,他引:7
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. 相似文献
5.
The Bacillus cytochrome P450 BM3 integrates an entire P450 system in one polypeptide and represents a convenient prokaryotic model for microsomal P450s. This self-sufficient class II P450 is also present in actinomycetes and fungi. By genome analysis we have identified additional homologues in the pathogenic species Bacillus anthracis and Bacillus cereus, and in Ralstonia metallidurans. This analysis also revealed a novel class of putative self-sufficient P450s, P450 PFOR, comprising a class I P450 that is related to Rhodococcus erythropolis CYP116, and a phthalate family oxygenase reductase (PFOR) module. P450 PFOR genes are found in a Rhodococcus strain, three pathogenic Burkholderia species and in the R. metallidurans strain that possesses a P450 BM3 homologue. Co-evolution of P450 and reductase domains is apparent in both types of self-sufficient enzymes. The new class of P450 enzymes is of potential interest for various biotechnological applications. 相似文献
6.
A J Green S L Rivers M Cheeseman G A Reid L G Quaroni I D Macdonald S K Chapman A W Munro 《Journal of biological inorganic chemistry》2001,6(5-6):523-533
The bioI gene has been sub-cloned and over-expressed in Escherichia coli, and the protein purified to homogeneity. The protein is a cytochrome P450, as indicated by its visible spectrum (low-spin haem iron Soret band at 419 nm) and by the characteristic carbon monoxide-induced shift of the Soret band to 448 nm in the reduced form. N-terminal amino acid sequencing and mass spectrometry indicate that the initiator methionine is removed from cytochrome P450 BioI and that the relative molecular mass is 44,732 Da, consistent with that deduced from the gene sequence. SDS-PAGE indicates that the protein is homogeneous after column chromatography on DE-52 and hydroxyapatite, followed by FPLC on a quaternary ammonium ion-exchange column (Q-Sepharose). The purified protein is of mixed spin-state by both electronic spectroscopy and by electron paramagnetic resonance [g values=2.41, 2.24 and 1.97/1.91 (low-spin) and 8.13, 5.92 and 3.47 (high-spin)]. Magnetic circular dichroism and electron paramagnetic resonance studies indicate that P450 BioI has a cysteine-ligated b-type haem iron and the near-IR magnetic circular dichroism band suggests strongly that the sixth ligand bound to the haem iron is water. Resonance Raman spectroscopy identifies vibrational signals typical of cytochrome P450, notably the oxidation state marker v4 at 1,373 cm(-1) (indicating ferric P450 haem) and the splitting of the spin-state marker v3 into two components (1,503 cm(-1) and 1,488 cm(-1)), indicating cytochrome P450 BioI to be a mixture of high- and low-spin forms. Fatty acids were found to bind to cytochrome P450 BioI, with myristic acid (Kd=4.18+/-0.26 microM) and pentadecanoic acid (Kd=3.58+/-0.54 microM) having highest affinity. The fatty acid analogue inhibitor 12-imidazolyldodecanoic acid bound extremely tightly (Kd<1 microM), again indicating strong affinity for fatty acid chains in the P450 active site. Catalytic activity was demonstrated by reconstituting the P450 with either a soluble form of human cytochrome P450 reductase, or a Bacillus subtilis ferredoxin and E. coli ferredoxin reductase. Substrate hydroxylation at the omega-terminal position was demonstrated by turnover of the chromophoric fatty acid para-nitrophenoxydodecanoic acid, and by separation of product from the reaction of P450 BioI with myristic acid. 相似文献
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Plant colonization by the vascular wilt fungus Fusarium oxysporum requires FOW1, a gene encoding a mitochondrial protein 总被引:2,自引:0,他引:2
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The soil-borne fungus Fusarium oxysporum causes vascular wilts of a wide variety of plant species by directly penetrating roots and colonizing the vascular tissue. The pathogenicity mutant B60 of the melon wilt pathogen F. oxysporum f. sp. melonis was isolated previously by restriction enzyme-mediated DNA integration mutagenesis. Molecular analysis of B60 identified the affected gene, designated FOW1, which encodes a protein with strong similarity to mitochondrial carrier proteins of yeast. Although the FOW1 insertional mutant and gene-targeted mutants showed normal growth and conidiation in culture, they showed markedly reduced virulence as a result of a defect in the ability to colonize the plant tissue. Mitochondrial import of Fow1 was verified using strains expressing the Fow1-green fluorescent protein fusion proteins. The FOW1-targeted mutants of the tomato wilt pathogen F. oxysporum f. sp. lycopersici also showed reduced virulence. These data strongly suggest that FOW1 encodes a mitochondrial carrier protein that is required specifically for colonization in the plant tissue by F. oxysporum. 相似文献
9.
Takaya N Kuwazaki S Adachi Y Suzuki S Kikuchi T Nakamura H Shiro Y Shoun H 《Journal of biochemistry》2003,133(4):461-465
Induction of the mitochondrial nitrate-respiration (denitrification) system of the fungus Fusarium oxysporum requires the supply of low levels of oxygen (O(2)). Here we show that O(2) and nitrate (NO(3)(-)) respiration function simultaneously in the mitochondria of fungal cells incubated under hypoxic, denitrifying conditions in which both O(2) and NO(3)(-) act as the terminal electron acceptors. The NO(3)(-) and nitrite (NO(2)(-)) reductases involved in fungal denitrification share the mitochondrial respiratory chain with cytochrome oxidase. F. oxysporum cytochrome c(549) can serve as an electron donor for both NO(2)(-) reductase and cytochrome oxidase. We are the first to demonstrate hybrid respiration in respiring eukaryotic mitochondria. 相似文献
11.
A new myosin motor-like chitin synthase gene, chsVb, has been identified in the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici. Phylogenetic analysis of the deduced amino acid sequence of the chsVb chitin synthase 2 domain (CS2) revealed that ChsVb belongs to class VII chitin synthases. The ChsVb myosin motor-like domain (MMD) is shorter than the MMD of class V chitin synthases and does not contain typical ATP-binding motifs. Targeted disrupted single (DeltachsVb) and double (DeltachsV DeltachsVb) mutants were unable to infect and colonize tomato plants or grow invasively on tomato fruit tissue. These strains were hypersensitive to compounds that interfere with fungal cell wall assembly, produced lemon-like shaped conidia, and showed swollen balloon-like structures in hyphal subapical regions, thickened walls, aberrant septa, and intrahyphal hyphae. Our results suggest that the chsVb gene is likely to function in polarized growth and confirm the critical importance of cell wall integrity in the complex infection process of this fungus. 相似文献
12.
Fischer M Knoll M Sirim D Wagner F Funke S Pleiss J 《Bioinformatics (Oxford, England)》2007,23(15):2015-2017
SUMMARY: The Cytochrome P450 Engineering Database (CYPED) has been designed to serve as a tool for a comprehensive and systematic comparison of protein sequences and structures within the vast and diverse family of cytochrome P450 monooxygenases (CYPs). The CYPED currently integrates sequence and structure data of 3911 and 25 proteins, respectively. Proteins are grouped into homologous families and superfamilies according to Nelson's classification. Nonclassified CYP sequences are assigned by similarity. Functionally relevant residues are annotated. The web accessible version contains multisequence alignments, phylogenetic trees and HMM profiles. The CYPED is regularly updated and supplies all data for download. Thus, it provides a valuable data source for phylogenetic analysis, investigation of sequence-function relationships and the design of CYPs with improved biochemical properties. Abbreviations: Cytochrome P450 Engineering Database, CYPED; cytochrome P450 monooxygenase, CYP; Hidden Markov Model, HMM. AVAILABILITY: www.cyped.uni-stuttgart.de 相似文献
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Cytochrome P-450scc. Cardiolipin as an effector of activity of a mitochondrial cytochrome P-450 总被引:2,自引:0,他引:2
J D Lambeth 《The Journal of biological chemistry》1981,256(10):4757-4762
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Kitazume T Takaya N Nakayama N Shoun H 《The Journal of biological chemistry》2000,275(50):39734-39740
The gene of a fatty-acid hydroxylase of the fungus Fusarium oxysporum (P450foxy) was cloned and expressed in yeast. The putative primary structure revealed the close relationship of P450foxy to the bacterial cytochrome P450BM3, a fused protein of cytochrome P450 and its reductase from Bacillus megaterium. The amino acid sequence identities of the P450 and P450 reductase domains of P450foxy were highest (40.6 and 35.3%, respectively) to the corresponding domains of P450BM3. Recombinant P450foxy expressed in yeast was catalytically and spectrally indistinguishable from the native protein, except most of the recombinant P450foxy was recovered in the soluble fraction of the yeast cells, in marked contrast to native P450foxy, which was exclusively recovered in the membrane fraction of the fungal cells. This difference implies that a post (or co)-translational mechanism functions in the fungal cells to target and bind the protein to the membrane. These results provide conclusive evidence that P450foxy is the eukaryotic counterpart of bacterial P450BM3, which evokes interest in the evolutionary aspects concerning the P450 superfamily along with its reducing systems. P450foxy was classified in the new family, CYP505. 相似文献
18.
Aberrant transposition of a Tc1-mariner element, impala, in the fungus Fusarium oxysporum 总被引:2,自引:0,他引:2
We previously determined that the impalaD transposable element of Fusarium oxysporum was able to mobilize a non autonomous copy of impala ( niaD::imp::hph), inserted in the niaD gene encoding nitrate reductase. Generally, mobilization results in the recovery of Nia(+) revertants at low frequency. In the course of this study, we recovered a transformant that gave rise to Nia(+) revertants at a high rate. These revertants displayed atypical phenotypes and showed a niaD hybridization pattern different from that in more typical revertants. Molecular analysis of the structure of the transformant and atypical revertants indicated that (i) in the transformant, two copies of impala, one defective and one active, were inserted at the same genomic locus in a head-to-head orientation; and (ii) all the revertants analyzed presented the same chromosomal rearrangement, an inversion resulting in the replacement of the niaD promoter by a new sequence containing a cryptic promoter. We also frequently observed additional DNA rearrangements (deletion or inversion) in these revertants. The sequences at the rearrangement junctions indicated the occurrence of a transposition event that used the ITRs (Inverted Terminal Repeats) of separate transposons arranged in direct orientation. These features can be interpreted as the consequences of an aberrant transposition process. Such a process may account for the rearrangements observed in some genomic regions containing multiple transposon ends, and could serve as a mechanism for the generation of genetic diversity. 相似文献
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The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b(5) (cyt b(5)) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b(5) molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 +/- 2 and 29 +/- 4.5 s(-1), respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of benzphetamine congruent with100-fold (k = 0.15 +/- 0.05 s(-1)) and cyclohexane congruent with10-fold (k = 2.5 +/- 0.35 s(-1)) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b(5), and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b(5) and CPR. Increasing the concentration of cyt b(5) enhanced the amount of product formed by cyt b(5) while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b(5):cyt P450 molar ratios examined, cyt b(5) inhibits the rate of NADPH consumption. Nevertheless, at low cyt b(5):cyt P450 molar ratios 相似文献