Mapping the primary structure of copper/topaquinone-containing methylamine oxidase fromAspergillus niger

The amino acid sequence of methylamine oxidase (MeAO) from the fungus Aspergillus niger was analyzed using mass spectrometry (MS). First, MeAO was characterized by an accurate molar mass of 72.4 kDa of the monomer measured using MALDI-TOF-MS and by a pI value of 5.8 determined by isoelectric focusing. MALDI-TOF-MS revealed a clear peptide mass fingerprint after tryptic digestion, which did not provide any relevant hit when searched against a nonredundant protein database and was different from that of A. niger amine oxidase AO-I. Tandem mass spectrometry with electrospray ionization coupled to liquid chromatography allowed unambiguous reading of six peptide sequences (11-19 amino acids) and seven sequence tags (4-15 amino acids), which were used for MS BLAST homology searching. MeAO was found to be largely homologous to a hypothetical protein AN7641.2 (EMBL/GenBank protein-accession code EAA61827) from Aspergillus nidulans FGSC A4 with a theoretical molar mass of 76.46 kDa and pI 6.14, which belongs to the superfamily of copper amine oxidases. The protein AN7641.2 is only little homologous to the amine oxidase AO-I (32% identity, 49 % similarity).     

The thioredoxin system of the filamentous fungus Aspergillus nidulans: impact on development and oxidative stress response

Redox regulation has been shown to be of increasing importance for many cellular processes. Here, redox homeostasis was addressed in Aspergillus nidulans, an important model organism for fundamental biological questions such as development, gene regulation or the regulation of the production of secondary metabolites. We describe the characterization of a thioredoxin system from the filamentous fungus A. nidulans. The A. nidulans thioredoxin A (AnTrxA) is an 11.6-kDa protein with a characteristic thioredoxin active site motif (WCGPC) encoded by the trxA gene. The corresponding thioredoxin reductase (AnTrxR), encoded by the trxR gene, represents a homodimeric flavoprotein with a native molecular mass of 72.2 kDa. When combined in vitro, the in Escherichia coli overproduced recombinant proteins AnTrxA and AnTrxR were able to reduce insulin and oxidized glutathione in an NADPH-dependent manner indicating that this in vitro redox system is functional. Moreover, we have created a thioredoxin A deletion strain that shows decreased growth, an increased catalase activity, and the inability to form reproductive structures like conidiophores or cleistothecia when cultivated under standard conditions. However, addition of GSH at low concentrations led to the development of sexual cleistothecia, whereas high GSH levels resulted in the formation of asexual conidiophores. Furthermore, by applying the principle of thioredoxin-affinity chromatography we identified several novel putative targets of thioredoxin A, including a hypothetical protein with peroxidase activity and an aldehyde dehydrogenase.     

The isopenicillin N acyltransferases of Aspergillus nidulans and Penicillium chrysogenum differ in their ability to maintain the 40-kDa alphabeta heterodimer in an undissociated form.

The isopenicillin N acyltransferases (IATs) of Aspergillus nidulans and Penicillium chrysogenum differed in their ability to maintain the 40-kDa proacyltransferase alphabeta heterodimer in an undissociated form. The native A. nidulans IAT exhibited a molecular mass of 40 kDa by gel filtration. The P. chrysogenum IAT showed a molecular mass of 29 kDa by gel filtration (corresponding to the beta subunit of the enzyme) but the undissociated 40-kDa heterodimer was never observed even in crude extracts. Heterologous expression experiments showed that the chromatographic behaviour of IAT was determined by the source of the penDE gene used in the expression experiments and not by the host itself. When the penDE gene of A. nidulans was expressed in P. chrysogenum npe6 and npe8 or in Acremonium chrysogenum, the IAT formed had a molecular mass of 40 kDa. On the other hand, when the penDE gene originating from P. chrysogenum was expressed in A. chrysogenum, the active IAT had a molecular mass of 29 kDa. The intronless form of the penDE gene cloned from an A. nidulans cDNA library and overexpressed in Escherichia coli formed the enzymatically active 40-kDa proIAT, which was not self-processed as shown by immunoblotting with antibodies to IAT. This 40-kDa protein remained unprocessed even when treated with A. nidulans crude extract. In contrast, the P. chrysogenum penDE intronless gene cloned from a cDNA library was expressed in E. coli, and the IAT was self-processed efficiently into its alpha (29 kDa) and beta (11 kDa) subunits. It is concluded that P. chrysogenum and A. nidulans differ in their ability to self-process their respective proIAT protein and to maintain the alpha and beta subunits as an undissociated heterodimer, probably because of the amino-acid sequence differences in the proIAT which affect the autocatalytic activity.     

The primary structure of thioredoxin from Chromatium vinosum determined by high-performance tandem mass spectrometry

The primary structure of thioredoxin, a redox protein isolated from Chromatium vinosum, was determined by high-performance tandem mass spectrometry, which permitted sequencing of the 14 peptides (ranging in length from 2 to 18 amino acids) generated by digestion with trypsin and of several peptides produced by Staphylococcus aureus protease. The mass spectrometrically determined molecular weights of the peptides from the latter digest were used to properly align the tryptic peptides, which could also be accomplished on the basis of the considerable homology with Escherichia coli thioredoxin. Finally, the molecular weight of the Chromatium thioredoxin was determined by mass spectrometry and found to be 11,748.0, in good agreement with 11,750.2 calculated for the proposed sequence. Although it was difficult to establish by mass spectrometry, five leucines and three isoleucines could be identified, leaving only eight undifferentiated.     

Mass spectrometrically derived amino acid sequence of thioredoxin from Chlorobium, an evolutionarily prominent photosynthetic bacterium

The amino acid sequence of the thioredoxin isolated from the photosynthetic green sulfur bacterium Chlorobium thiosulfatophilum was determined chiefly by fast atom bombardment mass spectrometry combined with Edman degradation and tandem mass spectrometry. For this purpose, the protein was digested with trypsin, alpha-chymotrypsin, thermolysin, and Staphylococcus aureus protease or combinations thereof. Chemical cleavage with cyanogen bromide was also used alone or in combination with trypsin. The resulting sequence of 108 amino acids is as follows: Ala-Gly- Lys-Tyr-Phe-Glu-Ala-Thr-Asp-Lys-Asn-Phe-Gln- Thr-Glu-Xle-Xle-Asp-Ser-Asp-Lys-(Ala-Val)-Xle- Val-Asp-Phe-Trp-Ala-Ser-Trp-Cys-Gly-(Pro-Cys)- Met-Met-Xle-Gly-Pro-Val-Xle-Glu-Gln-Xle-Ala-Asp- Asp-Tyr-Glu-Gly-Lys-Ala-Xle-Xle-Ala-Lys-Xle-Asn- Val-Asp-Glu-Asn-Pro-Asn-Xle-Ala-Gly-Gln-Tyr-Gly- Xle-Arg-Ser-Xle-Pro-Thr-Met-Xle-Xle-Xle-Ly s- (Gly-Gly-Lys)-Val-Val-Asp-Gln-Met-Val-Gly-Ala- Xle-Pro-Lys-Asn-Met-Xle-Ala-Lys-Lys-Xle-Asp-Glu-His-Il e-Gly (where Xle represents leucine or isoleucine; sequences in parentheses are based on homology considerations). It exhibits less than 53% homology with Escherichia coli thioredoxin.     

应用实时荧光PCR技术检测构巢曲霉的初步研究

目的 根据构巢曲霉（Aspergillus nidulans）3-磷酸甘油醛脱氢酶（Glyceraldehyde-3-phosphate dehydrogenase,GAPDH）基因特异位点设计并合成Taqman探针及引物,建立构巢曲霉实时荧光 PCR检测方法。方法 应用lasergene7.1软件对构巢曲霉与13种常见曲霉主要包括黑曲霉（A.niger）、烟曲霉（A.fumigatus）、杂色曲霉（A.versicolor）、土曲霉（A.terrus）、黄曲霉（A. flavus）、温特曲霉（A.wentii）、寄生曲霉（A. parasiticus）、泡盛曲霉（A.awamori）、米曲霉（A. oryzae ）、棒曲霉（A.cavatus）、赤曲霉（A.ruber ）、亮白曲霉（A.ochraceus）及赭曲霉（A.ochraceus）GAPDH基因序列比对分析,在特异位点设计引物和探针,建立构巢曲霉实时荧光 PCR检测方法,并对该方法进行特异性及敏感性分析。结果 用曲霉属22种41株不同曲霉及其他属的12株病原真菌验证实验表明,所建立的荧光PCR方法特异性强;检测灵敏度可达4.03×10－12μg/ml的模板DNA。 结论 应用实时荧光PCR技术能够有效检测构巢曲霉,该方法具有特异、灵敏、快速等特点,可在实际工作中应用。     

Thioredoxin is essential for photosynthetic growth. The thioredoxin m gene of Anacystis nidulans

We have taken advantage of the transformation properties of the cyanobacterium Anacystis nidulans R2 to investigate the importance of thioredoxin for photosynthetic growth. The gene encoding thioredoxin m, designated trxM, was cloned from A. nidulans using a synthetic oligonucleotide probe. Based on the nucleotide sequence, thioredoxin m of A. nidulans is composed of 107 amino acids and shares 84, 48, and 48% sequence identity with thioredoxins from Anabaena, spinach, and Escherichia coli, respectively. The trxM gene is single copy and is transcribed on a 510-nucleotide mRNA. We demonstrate that disruption of the trxM gene with a kanamycin resistance gene cartridge is a lethal mutation. Although dispensable in E. coli, thioredoxin is essential for the photosynthetic growth of A. nidulans.     

Amino acid sequence of thioredoxin isolated from rabbit bone marrow determined by tandem mass spectrometry

The amino acid sequence of the thioredoxin isolated from rabbit bone marrow was determined chiefly by high performance tandem mass spectrometry and fast atom bombardment mass spectrometry combined with manual Edman degradation. The sequences of peptides generated by digestion with trypsin alone or in combination with Staphylococcus aureus protease V8 or thermolysin were determined from their collision-induced dissociation mass spectra. Alignment of these sequences and additional sequence information were obtained from the collision-induced dissociation mass spectra of peptides obtained from digestion of the intact protein with S. aureus protease V8 and alpha-chymotrypsin. The resulting sequence of 104 residues is as follows: Val-Lys-Gln-Ile-Glu-Ser-Lys-Ser-Ala-Phe-Gln- Glu-Val-Leu-Asp-Ser-Ala-Gly-Asp-Lys-Leu-Val-Val- Val-Asp-Phe-Ser-Ala-Thr-Trp-Cys-Gly-Pro-Cys-Lys- Met-Ile-Lys-Pro-Phe-Phe-His-Ala-Leu-Ser-Glu-Lys- Phe-Asn-Asn-Val-Val-Phe-Ile-Glu-Val-Asp-Val-Asp- Asp-Cys-Lys-Asp-Ile-Ala-Ala-Glu-Cys-Glu-Val-Lys- Cys-Met-Pro-Thr-Phe-Gln-Phe-Phe-Lys-Lys- Gly-Gln-Lys-Val-Gly-Glu-Phe-Ser-Gly-Ala-Asn-Lys- Glu-Lys-Leu-Glu-Ala-Thr-Ile-Asn-Glu-Leu-Leu.     

2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans: characterization and comparison of both enzymes.

In Escherichia coli and Aspergillus nidulans, propionate is oxidized to pyruvate via the methylcitrate cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate is catalysed by 2-methylisocitrate lyase. The enzymes from both organisms were assayed with chemically synthesized threo-2-methylisocitrate; the erythro-diastereomer was not active. 2-Methylisocitrate lyase from E. coli corresponds to the PrpB protein of the prp operon involved in propionate oxidation. The purified enzyme has a molecular mass of approximately 32 kDa per subunit, which is lower than those of isocitrate lyases from bacterial sources ( approximately 48 kDa). 2-Methylisocitrate lyase from A. nidulans shows an apparent molecular mass of 66 kDa per subunit, almost equal to that of isocitrate lyase of the same organism. Both 2-methylisocitrate lyases have a native homotetrameric structure as identified by size-exclusion chromatography. The enzymes show no measurable activity with isocitrate. Starting from 250 mM pyruvate, 150 mM succinate and 10 microM PrpB, the enzymatically active stereoisomer could be synthesized in 1% yield. As revealed by chiral HPLC, the product consisted of a single enantiomer. This isomer is cleaved by 2-methylisocitrate lyases from A. nidulans and E. coli. The PrpB protein reacted with stoichiometric amounts of 3-bromopyruvate whereby the activity was lost and one amino-acid residue per subunit became modified, most likely a cysteine as shown for isocitrate lyase of E. coli. PrpB exhibits 34% sequence identity with carboxyphosphoenolpyruvate phosphonomutase from Streptomyces hygroscopicus, in which the essential cysteine residue is conserved.     

Saccharomyces cerevisiae centromere CEN11 does not induce chromosome instability when integrated into the Aspergillus nidulans genome.

We constructed Aspergillus nidulans transformation plasmids containing the A. nidulans argB+ gene and either containing or lacking centromeric DNA from Saccharomyces cerevisiae chromosome XI (CEN11). The plasmids transformed an argB Aspergillus strain to arginine independence at indistinguishable frequencies. Stable haploid transformants were obtained with both plasmids, and strains were identified in which the plasmids had integrated into chromosome III by homologous recombination at the argB locus. Plasmid DNA was recovered from a transformant containing CEN11, and the sequence of the essential portion of CEN11 was determined to be unaltered. The transformants were further characterized by using them to construct heterozygous diploids and then testing the diploids for preferential loss of the plasmid-containing chromosomes. The CEN11 sequence had little or no effect on chromosome stability. Thus, CEN11 does not prevent chromosomal integration of plasmid DNA and probably lacks centromere activity in Aspergillus spp.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe: highly homologous introns are inserted at the same position of the otherwise less conserved cox1 genes in Schizosaccharomyces pombe and Aspergillus nidulans.

The DNA sequence of the second intron in the mitochondrial gene for subunit 1 of cytochrome oxidase (cox1), and the 3'' part of the structural gene have been determined in Schizosaccharomyces pombe. Comparing the presumptive amino acid sequence of the 3'' regions of the cox1 genes in fungi reveals similarly large evolutionary distances between Aspergillus nidulans, Saccharomyces cerevisiae and S. pombe. The comparison of exon sequences also reveals a stretch of only low homology and of general size variation among the fungal and mammalian genes, close to the 3'' ends of the cox1 genes. The second intron in the cox1 gene of S. pombe contains an open reading frame, which is contiguous with the upstream exon and displays all characteristics common to class I introns. Three findings suggest a recent horizontal gene transfer of this intron from an Aspergillus type fungus to S. pombe. (i) The intron is inserted at exactly the same position of the cox1 gene, where an intron is also found in A. nidulans. (ii) Both introns contain the highest amino acid homology between the intronic unassigned reading frames of all fungi identified so far (70% identity over a stretch of 253 amino acids). However, in the most homologous region, a GC-rich sequence is inserted in the A. nidulans intron, flanked by two direct repeats of 5 bp. The 37-bp insert plus 5 bp of direct repeat amounts to an extra 42 bp in the A. nidulans intron. (iii) TGA codons are the preferred tryptophan codons compared with TGG in all mitochondrial protein coding sequences of fungi and mammalia.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Neurospora rca-1 gene complements an Aspergillus flbD sporulation mutant but has no identifiable role in Neurospora sporulation.

The Aspergillus nidulans flbD gene encodes a protein with a Myb-like DNA-binding domain that is proposed to act in concert with other developmental regulators to control initiation of conidiophore development. We have identified a Neurospora crassa gene called rca-1 (regulator of conidiation in Aspergillus) based on its sequence similarity to flbD. We found that N. crassa rca-1 can complement the conidiation defect of an A. nidulans flbD mutant and that induced expression of rca-1 caused conidiation in submerged A. nidulans cultures just as was previously observed for overexpression of flbD. Thus, the N. crassa gene appears to be a functional homologue of A. nidulans flbD and this is the first demonstration of functional complementation of an A. nidulans sporulation defect using a gene from an evolutionarily distant fungus. However, deletion of the rca-1 gene in N. crassa had no major effect on growth rate, macroconidiation, microconidiation, or ascospore formation. The only phenotype displayed by the rca-1 mutant was straight or counterclockwise hyphal growth rather than the clockwise spiral growth observed for wild type. Thus, if rca-1 is involved in N. crassa development, its role is subtle or redundant.