A novel peptide antibiotic, K‐582, which exhibited significant growth inhibition of Candida, viruses and ascites tumor in mice, was found in the culture medium of a strain of Metarhizium anisopliae by Kondo et al. (J. Antibiotics33, 535–542 (1980)]. K‐582 consisted of two components, designated K‐582 A and K‐582 B. Threonine, tyrosine, ornithine, and an unusual amino acid were common in both peptides, but lysine was an extra component of K‐582 A. The unusual amino acid was identified to be threo‐γ‐hydroxy‐L‐arginine (OHArg) by means of mass, nuclear magnetic resonance and infrared spectrometries of the derivatives and the related compounds. The threonine and the arginine were assigned to be L‐configuration, and the ornithine and the tyrosine to be D‐configuration in both K‐582 A and K‐582 B, and the lysine to be L‐configuration by comparison of their optical rotatory dispersion spectra with those of standard amino acids. The elucidation of primary structure revealed that they were closely related heptapeptides with the following sequence: K‐582 A:H‐Arg‐OHArg‐Orn‐Thr‐Orn‐Lys‐Tyr‐OH; K‐582 B:H‐Arg‐OHArg‐Orn‐Thr‐Orn‐OHArg‐Tyr‐OH, and had the identical sequence in terms of the configuration of their constituents, namely L‐L‐D‐L‐D‐L‐D.
A new basic peptide antibiotic designated as K-582 was isolated, purified and characterized. When K-582 was applied to a column of A1203 or Bio-Gel P-2 or CM Sephadex, two major peaks which were named Fraction I (K-582 A) and Fraction II (K-582 B) were obtained. The nitrogen content, the behavior in color reaction, the absorption bands of amide linkages in the infrared absorption spectrum, 1H NMR spectrum and C-13 NMR spectrum indicated the peptide nature of K-582 A and K-582 B. K-582 was effective against yeasts, but inactive against other Gram-positive bacteria, Gram-negative bacteria and Mycobacterium. The toxicity was low in mice.During our screening program for substances with antibiotic activity, we found a new substance named K-582 produced by the strain 582M which was isolated from a soil sample collected in Sendai and from the results of detailed taxonomical studies the strain was identified as Metarhizium anisopliae (METSCH.) SOROK. Var. anisopliae.The antibiotic, K-582, exhibited a significant growth inhibition of Candida in liquid media, of viruses in tissue culture and of ascites tumor in mice.This paper deals with the characterization of Metarhizium anisopliae (METSCH.) SOROK. var. anisopliae 582 M, the fermentation process, the isolation procedure and the properties of K-582. Taxonomical StudiesStrain 582 M was isolated by SHIGEJI KONDO from a soil sample collected in the vicinity of Sendai City, Miyagi Prefecture, Japan. The taxonomical characterization was carried out according to the methods described by BARNETT1), BARRON2) and TULLOCH3) and the strain 582 M was classified as Metarhizium anisopliae (METSCH.) SOROK. var. anisopliae. Macroscopic descriptionColonies on CZAPEK-peptone agar grew rapidly, attaining a diameter of 4.5 -5.0 cm in 2 weeks at 28°C. The colonies are more or less fasciculated, plane or sometimes lightly furrowed radially. They have white margins with olivaceous to dark olivaceous colored sporulating areas in center. Colonies are often intermixed or zonate. Transformation to light yellow to dark olivaceous colors occurs. Exudate is lacking. The odor was conspicuously pungent similar to that of streptomyces (Tables 1, 2 and 3.).
Formation of the basic antibiotic, K-582 was stimulated by supplying Metarrhiziumi anisopliae U-47 with several amino acids present in its structure. The addition of L-arginine to the basal medium resulted in the almost exclusive formation of K-582 B, while L-lysine increased K-582 A formation. Some carbon sources were observed to have effects similar to those obtained with the above mentioned amino acids. Furthermore, when L-arginine was added in excess to the basal medium, free)"-hydroxyarginine, which is a major constituent of the antibiotic, accumulated extra-and intra-cellularly. Free r-hydroxyarginine isolated from the culture broth of this microorganism was the threo-L-isomer. K-582 formation was repressed by glycerol, which exerted catabolite repression of i'hydroxyarginine synthesis. Imperfect fungus, Metarrhizium anisopliae (Metsch) Sorok var. anisopliae, produces the basic peptide antibiotics, K-582 A and B, which are excreted into the medium; those are effective against yeasts and certain viruses.') Their chemical structures were determined2:: K-582 A is Arg-Hyarg-Orn-Thr-Orn-Lys-Tyr; and K-582 B is Arg-Hyarg-Orn-Thr-Orn-Hyarg-Tyr*. r-Hydroxyarginine present in K-582 A and B, was isolated from a microbe for the first time, but has been known to be present in the sea cucumber, Polycheira rufescens3), the sea anemone, Anthopleura japonica Verrill4), and the seeds of 17 species of the genus Vicia5. Recently, MIZUSAKI and MAKISUMIB> synthesized chemically four stereoisomers of Hyarg via histidine. On the other hand, ITO-KAGAWA, one of the authors reported that in biogenesis of another peptide antibiotic, colistin, by Bacillus colistinus Koyama, yields increased markedly when amino acids of the aspartate family (isoleucine, threonine and a,r-diaminobutyric acid) were added." Specifically, a,rdiaminobutyric acid, a major constituent of the colistin molecule, stimulated colistin biosynthesis, and considerably inhibited the biosynthesis of cellular protein.') The present paper deals with the biogenesis of K-582 in relation to amino acid metabolism and, particularly, the role of Hyarg in the synthesis of K-582. In addition, free Hyarg, which accumulated extra-and intra-cellularly, was isolated and its chemical structure was elucidated. Methods Microorganism Metarrhizium anisopliae U-47, a mutant producing high yields of K-582 and Hyarg and derived from Metarrhizium anisopliae (Metsch) Sorok var. anisopliae strain No. 582M was used throughout this
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