Novobiocic acid

The biosynthesis of novobiocin has been reviewed 52, and the formation in cell-free extracts of novobiocic acid has been described 53. The latter can be formed from 3-am1no-4, 7-dihydroxy-8-methvl coumarin (B ring) and 4-hydroxy-3(3-methyl-2-butenyl)benzoic acid (A ring) by an enzyme that forms an amide bond between these precursors. Energy (ATP) is required but the mode of activation of the carboxyl group is unknown. Novobiocin itself possesses a sugar (noviose) but its biosynthesis and the formation of the glycosidic link are little studied 52. 

The end-product repression involves probably the blockage of formation of specific synthases, at the time of a maximum rate of secondary biosynthesis. This results in a gradual decline in the level of the enzyme and a retardation of the product formation. The phenomenon was observed in gramicidin S synthases by Bacillus brevis (30), bacitracin synthase in Bacillus licheniformis (31), anhydrotetracycline hydratase by treptomyces aureofaci-ens (4), novobiocic acid synthase in Streptomyces niveus (35) or dimethylallyltryptophan synthase by ciaviceps (33). 

Steffensky M, Li SM, Heide L (2000) Cloning, overexpression, purification of novobiocic acid synthetase from Streptomyces spheroides NCIMB 11891. J Biol Chem 275 21754-21760... 

Fermentation of Streptomyces niveus in the presence of Me-[ C]-L-methionine yielded novobiocin with an incorporation of 10 per cent of the added isotope °°. The radioactivity was distributed between the 0-methyl and gem-dimethyl groups of the sugar noviose (66 per cent) and the C-methyl group of the coumarin fragment (123,35 per cent) and thus indicated that the coumarin methyl group was formed by a process of C-methylation. The timing of this step is, however, not known. Enzyme systems have been isolated from Streptomyces niveus which are capable of rapid amide bond formation between the isoprenyl 4-hydroxybenzoic acid (125) and the coumarin (123) to give novobiocic acid. The reaction requires ATP and presumably involves some form of activation of the p-hydroxybenzoyl carboxyl function. 

Fig. 2. Suggested pathway for the synthesis of the noviose moiety of novobiocin Note R = novobiocic acid.

Fig. 4. The effect of precursors on the rate of novobiocin synthesis. Assay medium Glucose (30mg/ml) potassium phosphate buffer pH 7.5, 0.01 M 4-day washed cells and water -f the following additions at a concentration of 0.5 p.m/ml A. A ring -f B ring B. B ring -f 4-hydroxy benzoate C. 1. No additions 2. A ring 3. Bring 4. 4-hydroxy benzoate 5- novobiocic acid...

Addition of the B ring and 4-hy(h oxybenzoic acid again resulted in an increase by UV assay and in this case papergram analysis showed that all the material produced was novobiocin. The addition of 4-hydroxybenzoic acid alone or novobiocic acid had no effect on the rate of novobiocin synthesis. 

The formation of novobiocic acid but not novobiocin when the A and B rings are added indicates that the formation of the amide bond takes place rapidly and that this reaction precludes the formation of novobiocin from the added intermediates. This conclusion is further substantiated by the fact that the addition of novobiocic acid does not increase the rate of novobiocin synthesis, indicating that the attachment of the C ring does not occur. Therefore the glycosidic bond must be formed between the C and B rings prior to amide formation bond with the A ring. 

These data also indicate that 4-hydroxybenzoic acid is an intermediate or is converted readily to an intermediate in the synthesis of the A ring since it increases the rate of novobiocin formation when added with the B ring. Furthermore, this increased rate of novobiocin s5mthesis indicates that the syntheses of the A and B rings are the limiting reactions. The rates of their synthesis must be approximately equal since the A or B ring added singly does not result in the accumulation of novobiocic acid. 

Fig. 5. Formation of novobiocic acid by cell-free extracts of 5. niveus...

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