Studies on antibiotic biosynthesis by protoplasts and resting cells of Streptomyces echinatus. Part II. Effect of chromophore precursors.

Washed cell and protoplast suspensions from Streptomyces echinatus A8331, which produces the quinoxaline antibiotic echinomycin, have been used to study the effects of analogues of the natural chromophore upon antibiotic biosynthesis. Addition of quinoline-2-carboxylic acid caused a decrease in the labelling of echinomycin from L-[methyl-14C]methionine and an increase in labelled chloroform-extractable material. Quinoxaline-2-carboxylic acid increased the incorporation of radioactivity into both fractions. Thieno[3,2-b]pyridine-5-carboxylic acid, 6-methylquinoline-2-carboxylic acid, and quinoline-2-carboxylic acid (also to a lesser extent 7-chloroquinoxaline-2-carboxylic acid) increased markedly the incorporation of radioactivity into chloroform-extractable material and virtually abolished echinomycin synthesis. Autoradiographs of extracts from suspensions supplemented with the latter four analogues revealed bis-substituted metabolites not found in unsupplemented cultures. When protoplast suspensions were incubated with L-[U-14C]serine, L-[U-14C]valine, or DL-[benzene ring-U-14C]tryptophan, quinoline-2-carboxylic acid, thieno[3,2-b]pyridine-5-carboxylic acid, and 6-methylquinoline-2-carboxylic acid directed the synthesis of antibiotically active bis derivatives at the expense of echinomycin. When analogues of quinoxaline-2-carboxylic acid previously found unsuitable for incorporation by growing cultures were tested in protoplast suspensions, only isoquinoline-3-carboxylic acid caused a large increase in the incorporation of radioactivity from L-[methyl-14C]methionine into chloroform-extractable material. With DL-[benzene ring-U-14C]tryptophan as the radiolabel, benzotriazoline-2-acetic acid and 6-bromoquinoxaline-2-carboxylic acid as well as isoquinoline-3-carboxylic acid sharply reduced the labelling of echinomycin.

Stereochemistry of beta-, gamma-, and epsilon-ring formation in bacterial C50.

Cell-free systems from Corynebacterium poinsettiae and Micrococcus luteus incorporated labeled mevalonic acids into acyclic C40 and cyclic C50 carotenoids. When (3R,4R)-[2-14C,4-3H1]mevalonate was used as substrate, the 14C:3H ratios of C.p.450 and sarcinaxanthin showed that the hydrogen atoms at C-2 of both carotenoids, and that at C-6 of sarcinaxanthin, are derived from the 4-pro-R position of mevalonate. The 14C:3H ratios of C.p.450 and sarcinaxanthin synthesized from (2RS,3R)-[2-14C,2-3H2]mevalonate showed that both hydrogen atoms of C-4 are derived from those at C-2 of mevalonate. These results exclude epsilon- and beta-rings as precursors of the gamma-ring. They also exclude the interconversion of the epsilon- and beta-rings. Sarcinaxanthin samples synthesized from (3R,4R)-[2-14C,4-3H1]- and (2RS,3R)-[2-14C,2-3H2]mevalonate by a cell-free system from M. luteus were found to undergo isomerization in strong alkali. The major product of isomerization (85%) was decaprenoxanthin (epsilon-ring) with the beta-ring C.p.450 present in small amounts (3% yield). The 14C:3H ratios of these isomerization products were consistent with the loss of one C-4 hydrogen atom from each epsilon-ring of the former and one C-6 hydrogen atom from each beta-ring of the latter.

Stereochemistry of allene biosynthesis and the formation of the acetylenic carotenoid diadinoxanthin and peridinin (C37) from neoxanthin.

Intact cells of the alga Amphidinium carterae (Dinophyceae), and a cell-free system prepared from it, incorporated 14C, 3H-labelled mevalonate into lycopene, beta, beta-carotene, zeaxanthin, neoxanthin, diadinoxanthin and peridinin. The 14C/3H ratios of zeaxanthin, neoxanthin and diadinoxanthin formed from (2RS,3R)-[2-14C,2-3H2]mevalonate show that a hydrogen atom from C-2 of mevalonate is retained in the allene at C-8, and also at C-12 of peridinin. (3R,4R + 3S,4S)-[2-14C,4-3H1]Mevalonate gave 14C/3H ratios in peridinin which show that C-14 is lost. The three carbon atoms excised during the formation of the C37 carotenoid peridinin are C-13, C-14 and C-20 of neoxanthin.

Retention of the 4-pro-R hydrogen atom of mevalonate at C-2,2' of bacterioruberin in Halobacterium halobium.

Intact cells of Halobacterium halobium fed with (3R,4R)-[2-14C,4-3H1]mevalonic acid were found to incorporate label into acyclic C40 and C50 carotenoids, of which bacterioruberin was the most abundant. The 14C/3H ratios of the isolated carotenoids demonstrated that the 4-pro-R hydrogen atom of mevalonic acid was retained at the C-2 and C-2' positions of bacterioruberin. Diphenylamine was found to inhibit the production of bacterioruberin.