A nylon-degrading enzyme found in the extracellular medium of a ligninolytic culture of the white rot fungus strain IZU-154 was purified by ion-exchange chromatography, gel filtration chromatography, and hydrophobic chromatography. The characteristics of the purified protein (i.e., molecular weight, absorption spectrum, and requirements for 2,6-dimethoxyphenol oxidation) were identical to those of manganese peroxidase, which was previously characterized as a key enzyme in the ligninolytic systems of many white rot fungi, and this result led us to conclude that nylon degradation is catalyzed by manganese peroxidase. However, the reaction mechanism for nylon degradation differed significantly from the reaction mechanism reported for manganese peroxidase. The nylon-degrading activity did not depend on exogenous H2O2 but nevertheless was inhibited by catalase, and superoxide dismutase inhibited the nylon-degrading activity strongly. These features are identical to those of the peroxidase-oxidase reaction catalyzed by horseradish peroxidase. In addition, α-hydroxy acids which are known to accelerate the manganese peroxidase reaction inhibited the nylon-degrading activity strongly. Degradation of nylon-6 fiber was also investigated. Drastic and regular erosion in the nylon surface was observed, suggesting that nylon is degraded to soluble oligomers and that nylon is degraded selectively.

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The A chain of ricin (RA) is a cytotoxic RNA N-glycosidase that inactivates ribosomes by depurination of the adenosine residue at position 4324 in 28S rRNA. Of the 267 amino acids in the protein, 231 could be deleted in one or another of 83 mutants, without the loss of the capacity to catalyze hydrolysis of a single specific nucleotide in rRNA [Morris, K. N. & Wool, I. G. (1992) Proc. Natl Acad. Sci. USA 89, 4869Ϫ4873]. Expression of 29 selected deletion mutants of RA in prokaryotic cell-free coupled transcription-translation reactions was carried out and the activities of the mutants were assessed by monitoring depurination of reticulocyte ribosomes. Kinetic analysis of five deletion mutants which retained detectable activity was performed. Deletion of amino acids outside the putative active-site cleft in these mutants significantly affected the catalytic rate rather than the interaction with ribosomes. From these data, the amino acids far from the active-site cleft appeared to be involved in alignment of the key residues for catalysis and interaction with the target tetraloop structure of 28S rRNA.Keywords : ricin; N-glycosidase; cell-free coupled transcription-translation; ribosome; protein synthesis.Ricin is a cytotoxic RNA N-glycosidase that is found in the seeds of Ricinus communis. Proricin is processed by removal of a connecting peptide of 12 amino acids to form an A chain of 267 residues and a B chain of 262 residues linked by a disulfide bond [1]. The toxic ricin A chain (RA) inhibits protein synthesis by inactivating ribosomes ; the inhibition is the result of the hydrolysis of the bond between the base and the ribose of the adenosine at position 4324 (A4324) in 28S rRNA [2,3]. RA is extraordinarily toxic; and a single molecule will inactivate 1500 ribosomes min Ϫ1 ; indeed, a single molecule is sufficient to kill a cell [1] and this one covalent modification accounts entirely for the cytotoxicity.The value of an analysis of the mechanism of action of ribotoxins is that it directs attention to components of the ribosome where efforts to comprehend functional correlates of the structure are likely to be rewarded. That this one RA-catalyzed depurination inactivates the ribosome implies that this region of 28S rRNA is crucial for the function of the particle. There is evidence that the ricin domain is involved in elongation-factor-1-(or Tu)-dependent binding of aminoacyl-tRNA to the ribosomal A site and elongation-factor-2-(or G)-catalyzed GTP hydrolysis and translocation of peptidyl-tRNA to the P site [4].A substantial effort has been made to relate the structure of RA to its mechanism of action spurred in part by the use of the protein in the construction of immunotoxins for cancer therapy [5]. The amino acid sequences of ricin and of several homologous proteins have been determined [6Ϫ12] and an atomic structure of ricin has been obtained from X-ray diffraction of crystals [13Ϫ15]. In the three-dimensional structure, there is a prominent cleft that was proposed to be the active site of RA before the ...

Four isozymes of manganese peroxidase (MnP) were identified in the culture fluid of the hyperlignolytic fungus IZU-154 under nitrogen starvation conditions. One of them was purified and characterized kinetically. The specific activity and k cat /K m value of the MnP from IZU-154 were 1.6 times higher than those of the MnP from a typical lignin-degrading fungus, Phanerochaete chrysosporium. Two cDNAs encoding MnP isozymes from IZU-154 were isolated. The coding sequence of the two cDNAs, IZ-MnP1 cDNA and IZ-MnP2 cDNA, were 1,152 (384 amino acids) and 1,155 (385 amino acids) bp in length, respectively. They exhibit 96.2% identity at the nucleotide level and 95.1% identity at the amino acid level. Southern blot analysis indicated that two MnP isozyme genes exist in IZU-154 genomic DNA. The primary structures of two MnPs from IZU-154 were similar to those of MnPs from P. chrysosporium. The amino acid sequences including the important residues identified in MnPs from P. chrysosporium, such as the manganese-binding residues, the calcium-binding residues, the disulfide bonds, and the N-glycosylation site, were conserved in the two deduced IZ-MnPs. However, several discrepancies were found in the context around the distal histidine residue between MnP from IZU-154 and MnP from P. chrysosporium, which likely led to the difference in the kinetic parameters for MnP function.