Cyclopenine biosynthesis

Preliminary results indicate that a cyclic compound related to those involved in cyclopenin biosynthesis (see above) could be involved in anthramycin biosynthesis, with modification of the tyrosine moiety taking place after formation of this cyclic compound. 

Significant progress has been made in the identification of enzymes from P. cyclopium which catalyse the various reactions of cyclopenin (124) and cyclopenol (125) biosynthesis.105 One of these is cyclopeptine dehydrogenase which catalyses the interconversion of (122) and (123).105a,1°6 Further research107 has shown that... 

The first correct structures of benzodiazepine alkaloids, namely, those of cyclopenin (3) and cyclopenol (4), were published in 1963 by Mohammed and Luckner (47). In 1965 the structure of the antibiotic anthramycin (8) was established (28), followed by tomaymycin (12) and oxotomaymycin (13) in 1971 (26). The biosynthetic pathway to both the cyclopenin group and the anthramycin-tomaymycin group of alkaloids was elucidated in the following years and has been included in previous reviews (32,33). Later additions to the small list of naturally occurring benzodiazepines comprise aszonalenin (6) (16), asperlicin (7) (31), and auranthine (5) (85). The biosynthesis of the latter alkaloids has not yet been investigated in detail. 

These findings prove the pathway of cyclopenin-cyclopenol biosynthesis shown in Fig. 5. In the first step, the cyclic dipeptide cyclopeptine (1) is formed... 

Fig. 10. In vitro activities of the enzymes of alkaloid biosynthesis and rates of alkaloid formation in hyphae of Penicitlium cyclopium (75). Cultivation conditions were as described in the legend to Fig. 9. At the times indicated by arrows, cycloheximide (100 p.g/ml) was added to the culture medium. All values are in units per 1 cm of culture area. (A) Anthranilate adenylyltransferase (AA) (100 = 5.6 pkat) ( ) cyclopeptine dehydrogenase (CD) (100 = 40 pkat) ( ) dehydrocyclopeptine epoxidase (DE) (100 = 0.42 pkat) (A) cyclopenin m-hydroxylase (CH) (100 = 12 pkat) (O) cyclopenin-cyclopenol formation in vivo (100 = 9 pmol/sec).

Fig. 12. Metabolic pathways leading to the biosynthesis of phenylalanine, anthranilic acid, and cyclopenin in Penicillium cyclopium (67). Symbols in parenthesis indicate observed feedback inhibition (-) or activation (-I-) of enzyme activities by L-amino acids. Number in circles (pkat/cm of mycelial area) represent either in vitro activities of the enzymes indicated or the rate of alkaloid formation in vivo. All data were measured after 7 days of growth in surface cultures. E 4-P, Erythrose 4-phosphate PEP, phosphoenolpyruvate DAHP, 3-deoxo-D-arabiuoheptuIosonic acid-7-phosphate InGP, inositol glycerophosphate SAM, 5-adeno ylmethionine.

Fig. 14. Overview of regulatory mechanisms acting at the level of transport and channeling of the alkaloid precursor phenylalanine in Penicillium cyclopium (60). (1) Under the influence of P-factor, the biosynthesis of vacuolar phenylalanine carriers is stimulated. (2) Above a threshold concentration, cellular methionine and cysteine inactivate vacuolar phenylalanine carriers. (3) Distinct concentrations of cellular ATP inhibit the efflux from the vacuole high levels of cytosolic amino acids stimulate efflux in the presence of sufficient ATP. (4) The vacuolar phenylalanine pool is most probably involved in triggering the expression of alkaloid metabolism. (5) In the idiophase, cyclopenin stimulates enzymes involved in the biosynthesis of phenylalanine.

Benzodiazepine Alkaloids.—Cyclopenin (49) and cyclopenol (50) are isolated from Penicillium cyclopium. Their biosynthesis would appear to involve one molecule of phenylalanine and one of anthranilic acid and indeed the biosynthetic pathway to viridicatin and viridicatinol in Penicillium viridicatum from these two amino-acids has (49) and (50) as intermediates. ... 

The failure of the cyc/o-alanyltyrosyls to act as mycelianamide precursors contrasts with the observed incorporation of the cyclic dipeptide (161) into brevianamide A (160) in P. brevicompactum and similar results with the related metabolite echinulin. This difference was attributed to differences in the number of reactions which take place on an enzyme complex before exchange can occur with unbound material.It may be noted that the early stages in the biosynthesis of cyclopenin and cyclopenol, which are also modified cyclic dipeptides, appear to involve enzyme-bound intermediates. ... 

Benzodiazepine Aikaioids.—Benzodiazepine alkaloids, e.g. cyclopenin (123), are produced by Penicillium cyclopium. Their biosynthesis is pretty well understood. Recently, the production of enzymes involved in the biosynthesis and formation of alkaloid in relation to the development of the organism has been studied. The uptake of one of the precursors, i.e. phenylalanine, has also been studied. ... 

Nover, M. Luckner, On the biosynthesis of cyclopenin and cyclopenol, benzodiazepine alkaloids from Penicillium cyclopium Westling, Europ. J. Biochem. 10, 268 - 273 (1969). 

The enzymes involved in the alkaloid metabolism of P. ovoloplum become measurable during the transition from tne trophophase to the idiophase. The activity increase of cyclopeptine dehydrogenase, dehydrocyclopeptine ep-oxidase and cyclopenin m-hydioxylase is inhibited by 5-fluorouracil and cycloheximide, indicating that it depends on RNA and protein biosynthesis (7 - 9). Hence like many other secondary metabolic activities (cf, 2,... 

Fig. 3 In vitro activities of the enzymes of alkaloid biosynthesis and rates of cyclopenin-cyolopenol formation in vivo in hyphae of P. cycloplum (Prom 8, 9)...

In contrast to the hyphal cells, which after their synthesis specialize with formation of the means necessary for alkaloid metabolism, with the conidiospores specialized cells are formed de novo. The conidia contain the enzymes of cyclopenin-cyclopenol biosynthesis as constitutive proteins (Fig. 5), Hence these enzymes are synthesized during cell division in the conidia-pro-ducing cells, the phialides. However, as with alkaloid biosynthesis in the hyphae in spite of optimum enzyme... 

Fig. 6. In vitro activities of the enzymes of cyclopenin-viridicatin biosynthesis and rates of the formation of the alkaloids cyclopenin and cyclopenol in vivo during ripening of the conidiospores of Penicillium cyclopium...

Enzymes of alkaloid biosynthesis are induced in a coordinated manner by an internal signal during development of the mold. However, the rates of alkaloid production increase later than enzyme activities, The increase of both processes can be stopped by addition of inhibitors of gene expression, e.g., 6-fluorouracil or cycloheximide (cf. arrows), indicating that alkaloid production is controlled by the formation of a protein which is not an enzyme of alkaloid biosynthesis. Ordinate relative values A A anthranilic acid adenylyl transferase (a) CD cyclopeptine dehydrogenase (v) DE dehydrocyclopeptine epoxidase ( ) CmH cyclopenin m-hydroxvlase (A)... 

Typical examples are experiments in which COg was administered to intact leaves in competition with mevalonic acid in the location of the site of plastid quinone biosynthesis and experiments with Penicillium cyclopium, where the channeling of the precursor L-phenylalanine and its incorporation into the alkaloids of the cyclopenin-viridicatin group and proteins were studied... 

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