RESUMO
Alamethicin synthetase was extracted from the fungus Trichoderma viride at the end of its exponential growth phase. It is multienzyme complex with a molecular weight of approx. 480 000. The biosynthesis of alamethicin is initiated on the synthetase by acetylation of thiolester-bound aminoisobutyric acid, which remains enzyme bound. Acetyl-CoA serves as the acetate donor. Of the alamethicin constituents, glycine, alanine and valine are also acetylated when incubated alone. This acetylation is prevented by added aminoisobutyric acid, which indicates that the site on alamethicin synthetase catalyzing the acetylation has a preference for aminoisobutyric acid. Alamethicin formation on the synthetase is terminated by linkage of phenylalaninol to the carboxyl terminus of the peptide. It is unlikely that the amino alcohol is a degradation product of alamethicin or that it had been split off from the synthetase complex. Thus it is probably the reaction product of a separate enzyme system.
Assuntos
Alameticina/biossíntese , Antibacterianos/biossíntese , Fungos Mitospóricos/metabolismo , Peptídeo Sintases/metabolismo , Trichoderma/metabolismo , Acetilação , Alameticina/síntese química , Ácidos Aminoisobutíricos/metabolismo , Sítios de Ligação , Fenômenos Químicos , Química , Fenilalanina/análogos & derivados , Fenilalanina/metabolismoRESUMO
The sequence of a peptide, trichokonin-II (TK-II), obtained from the culture broth of Trichoderma koningii OUDEMANS, was examined by ion-spray ionization mass spectrometry (ISI-MS), including the collision-induced dissociation (CID) technique. TK-II was concluded to be a mixture of three peptaibols, TK-IIa, TK-IIb, and TK-IIc.
Assuntos
Alameticina/análogos & derivados , Trichoderma/química , Alameticina/biossíntese , Alameticina/química , Sequência de Aminoácidos , Meios de Cultura/análise , Espectrometria de Massas , Dados de Sequência Molecular , Trichoderma/metabolismoRESUMO
The production of polypeptides containing a high percentage of 2-methylalanine residues by a number of isolates of Trichoderma spp. has been examined. It has been shown that good yields (0.5-1.0 g L-1) can be achieved on synthetic media provided an insoluble carbohydrate is included and provided single-spore isolates that have this production ability are selected from time to time. Such yields could not be obtained on any single nitrogen source investigated, but a mixture of potassium nitrate, glutamine, and 2-methylalanine was effective. It was shown that at least eight polypeptides were produced in shake-flask or tank fermentation and that the proportions of these metabolites depended on the fermentation temperature, its pH, age, and aeration. Fermentation conditions for enhancing the production (independently) of two of the metabolites at the expense of the others are given. These two metabolites have been obtained in crystalline form and details of some of their physical and chemical properties are given.
Assuntos
Alameticina/biossíntese , Antibacterianos/biossíntese , Fungos Mitospóricos/metabolismo , Trichoderma/metabolismo , Alameticina/análise , Alameticina/isolamento & purificação , Fenômenos Químicos , Química , Cromatografia Líquida de Alta Pressão , Meios de Cultura , Fermentação , Concentração de Íons de Hidrogênio , Nitrogênio/metabolismo , TemperaturaRESUMO
The biosynthesis of the cyclic octadecapeptide, alamethicin, in a cell-free system of Trichoderma viride has been investigated. It was shown that nucleic acid- and ribo-some-free extracts of Trichoderma viride could catalyze alamethicin biosynthesis. Puromycin, erythromycin and RNAse did not inhibit this synthesis. The Sephadex G 200 filtrate contains a fraction (Kav=0.1) that catalyzes the biosynthesis of alamethicin and shows an ATP-32PPi exchange with 6 of the 8 constituent amino acids of alamethicin. The activated amino acids are bound to the enzyme as aminoacyl adenylates and as thiolesters in a proportion of 1 : 1. About 50% of each bound amino acid could be split off with 7% TCA. The TCA-stable bound amino acid could be split by mercury acetate, hydroxylamine and performic acid. N-ethylmaleimide blocked the binding of 50% of the amino acids to the enzyme, proving that some of the amino acids first bound as aminoacyl adenylates are then transferred into a thiolester bond.