Isolation of a new antibiotic, alaremycin, structurally related to 5-aminolevulinic acid from Streptomyces sp. A012304.

A new antibiotic, which is structurally related to 5-aminolevulinic acid, a precursor of heme biosynthesis, and named alaremycin, was isolated from the culture broth of an actinomycete strain through a random screening with the blue assay to detect the formation of anucleate cells in Escherichia coli. The producing strain was identified as Streptomyces sp. by morphological, physiological, chemical and genetic criteria. Alaremycin was purified from the culture supernatant by HP-20 hydrophobic-interaction chromatography, sequential solvent/water extraction in the acidic or alkaline pH range, and QMA cation-exchange chromatography. The chemical structure of alaremycin was determined as 5-acetamido-4-oxo-5-hexenoic acid by analyses of mass and NMR spectra. The antibacterial activity of alaremycin was enhanced in the presence of 5-aminolevulinic acid.

beta-methylnorleucine, an antimetabolite produced by Serratia marcescens.

An amino acid was formed by alpha-aminobutyrate-resistant mutants of Serratia marcescens grown in a medium containing norvaline. This amino acid was identified as erythro-beta-methyl-L-norleucine [(2S,3S)-2-amino-3-methylhexanoic acid] by instrumental analyses. beta-Methylnorleucine inhibited the growth of several bacteria in synthetic medium.

Enzymes involved in 3,5-diaminohexanoate degradation by Brevibacterium sp.

Cell-free extracts of Brevibacterium sp. L5 grown on DL-erythro-3,5-diaminohexanoate were found to contain a 3-keto-5-aminohexanoate cleavage enzyme that converts 3-keto-5-aminohexanoate and acetyl-coenzyme A (CokA) to 3-aminobutyryl-CoA and acetoacetate and a deaminase that coverts L-3-aminobutyryl-CoA to crotonyl-CoA. The cleavage enzyme has been purified extensively, and some of its properties have been determined for comparison with the 3-keto-6-acetamido-hexanoate cleavage enzyme of Pseudomonas sp. B4. The deaminase has been partially purified and characterized. Both the cleavage enzyme and the deaminase are induced by growth on 3,5-diaminohexanoate. The presence of these and other accessory enzymes in Brevibacterium sp. extracts accounts for the results of earlier tracer experiments which showed that C-1 and C-2 of 3-keto-5-aminohexanoate are converted mainly to acetoacetate and acetate, whereas C-3 to C-6 are converted mainly to 3-hydroxybutyrate or its coenzyme A thiolester. The enzymes observed in extracts of Brevibacterium sp. can account for the conversion of 3,5-diaminohexanoate to acetyl-CoA.

2-Amino-5-methyl-5-hexenoic acid, a methionine analog produced by Streptomyces sp. MF374-C4.

2-Amino-5-methyl-5-hexenoic acid (AMHA), a new methionine analog, was isolated from a fermentation broth of Streptomyces sp. MF374-C4 based on its reversal of the effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in a test system that determines the size of growth zones of revertants (His+) of Salmonella typhimurium TA1535. AMHA also inhibited growth of the tester strain in a synthetic medium. These AMHA activities were abolished by methionine. The incidence of spontaneous streptomycin-resistant mutations of Escherichia coli K12 was not decreased by AMHA at concentrations where cell growth was partially inhibited. AMHA inhibited protein synthesis but not DNA or RNA synthesis in S. typhimurium TA1535 and E. coli K-12. The analog inhibited formation of methionyl-tRNA but not of valyl-tRNA in a cell-free system of E. coli, and supported ATP-PPi exchange in the cell-free system. At concentrations where it inhibited cell growth, AMHA decreased the number of foci, induced by ROUS sarcoma virus, on cultured sheets of chick-embryo fibroblasts. The effects of AMHA on focus formation and on the cell growth were overcome by methionine.

Beta-hydroxynorleucine: separation of its isomers and biological studies.

Separation of the four isomers of beta-hydroxynorleucine was accomplished by partition column chromatography and asymmetric enzymatic hydrolysis of the N-chloroacetyl derivatives. From these, the corresponding N-chloroacetyl derivatives were made. The purity and configuration of each isomer of the free acid and N-chloroacetylated derivative were ascertained by: (a) paper chromatography in five solvent systems, (b) elemental analysis, (c) Van Slyke nitrous acid determination of alpha-carbonyl carbon, and (d) Van Slyke ninhydrin determination of alpha-carbonyl carbon, and (e) optical rotation. Comparison of the rate of enzymatic hydrolysis by hog renal acylase I of the N-chloroacetyl derivative of the L-isomers of each diastereomer showed that the acyl B isomer is a better substrate than the acyl A isomer, where A denotes the faster moving diastereomer and B denotes the slower moving diastereomer in a defined chromatographic solvent system. Microbiological assay using Lactobacillus casei in a system selected for screening for possible antitumor activity indicated that while none of the isomers as free amino acids had any growth inhibitory action, the N-acylated isomers showed modest but significant activity. The N-chloroacetyl derivative of the D-enantiomorph of diastereomer B exhibited the greatest growth inhibitory activity, showing about twice the activity of the other three isomers.