6-methylsalicylate synthetase

Few areas of natural products chemistry have seen as many major advances in the study of biosynthetic pathways as have occurred in polyketide compounds. Birch and Donovan (1953) demonstrated that a wide range of structural types are derived from acetate (later shown to be acetate and malonate). In experiments witfi deuterated precursors, acetate serves preferentially as a starter unit for the formation of 6-methylsalicylic acid in Penicillium griseofulvum (Simpson, 1983). Thus, polyketides are derived from the same precursors as fatty acids and the initial step seems to be similar (Fig. 5.1). Extensive purifrcation of 6-methylsalicylate synthetase from Penicillium patulum has been performed. This enzyme system is distinct and separable from the co-occurring fatty acid synthetase and has a molecular weight approximately half that of the former enzyme. NADPH is required as a coenzyme for methylsalicylate synthetase from this source (O Hagan, 1990 Packter, 1980). 

Formation of 6-methylsalicylic acid involves condensation of an acetyl-CoA unit with three units of malonyl-CoA. The entire sequence is enzyme bound and intermediates are not released. Furthermore, attempts to introduce exogenous intermediates in feeding studies failed. As mentioned above, extensive purification of 6-methylsalicylate synthetase from Penicillium patulum yielded an enzyme complex distinct and separable from fatty acid synthetase and approximately half in molecular weight. NADPH is required for production of 6-methylsalicylic acid (Packter, 1980). The application of NMR spectroscopy to study of the biosynthetic steps in the formation of 6-methylsalicylic acid and other polyketides has been reviewed (Simpson, 1987). 

Since fatty acid and aromatic synthetases have many properties in common, it is interesting to consider the effect of omitting NADPH from incubations catalyzed by these enzymes and to examine the consequences. In all cases studied from animal (Bressler and Wakil, 1%2 Nixon et al., 1%8), yeast (Yalpani et al., 1969), and bacterial origin (Brock and Bloch, 1966), fatty acid synthesis is naturally abolished, but a Q compound, triacetic acid lactone (4-hydroxy-6-methyl-2-pyrone) (VII), is formed instead. This product is also made when the NADPH-requiring 6-methylsalicylate synthetase is deprived of this nucleotide (Dimroth et al., 1972), but the relative rate of synthesis is considerably greater than that produced by fatty acid synthetase in P. patulum (Yalpani et al., 1%9). However, Scott et al. (1971) have reported that triacetic acid lactone is also formed by 6-methylsalicylate synthetase, albeit at a reduced rate, even in the presence of NADPH. 

Scheme 4. Mechanism for the release of triacetic acid lactone (VII). Enz, Fatty acid or 6-methylsalicylate synthetase HS —, phosphopantetheine binding site.

For more complex metabolites (phenols and tetraacetic acid lactone), further condensation of the triacetyl residue must take place and can only proceed if the priming acetyl residue, with its C-5 oxygen function, is stabilized away from the thioester region of the acyl-enzyme intermediate at this time. Possibly, the presence of NADPH assists in this process, in appropriate enzymes, since the ability to form triacetic acid lactone has been demonstrated by fatty acid and 6-methylsalicylate synthetases when deprived of this nucleotide, but other synthetases have not been tested for this effect. Moreover, the analogous formation of the styrylpyrone bisnoryangonin by flavan-one synthase (Kreuzaler and Hahlbrock, 1975a,b) confirms that the potential for synthesis of stable products with shorter chains does exist. 

Extensive purification of 6-methylsalicylate synthetase from Penicil-lium patulum has been carried out. It is distinct from fatty acid synthetase, separable from it, and of half the molecular weight both enzymes are complexes of several enzymes. One molecule of 6-methylsalicylic acid is generated by 6-methylsalicylate synthetase from one molecule of acetyl-CoA and three of malonyl-CoA in the presence of one molecule of NADPH. as coenzyme no free intermediates can be detected. In the absence of NADPH, triacetic acid lactone 3.15) is formed as the sole product. The same lactone is... 

F. Lynen, H. Engeser, J. Friedrich, W. Schindlbeck, R. Seyffert, and F. Wieland, Fatty acid synthetase of yeast and 6-methylsalicylate synthetase of Penicillium patulum - two multienzyme complexes, iji "Microenvironments and Metabolic Conpartmentation," P.A. Srere and R.W. Estabrook, eds.. Academic Press, New York (1978). 

A soluble extract was obtained by Lynen and Tada (1961) fromP. patulum that required acetyl-CoA and malonyl-CoA plus NADPH for activity synthesis of 6-methylsalicylic acid, therefore, involves a reductive step to remove an oxygen function. These substrates are identical to those encountered in fatty acid synthesis, and this has led to the proposal that a multienzyme complex similar to fatty acid synthetase might be involved (Lynen and Tada, 1961 Lynen, 1961). Active thiol sites associated with this enzyme were also implicated in binding the intermediates by thioester linkage. The general mechanism applicable to the two synthetases, therefore, appears to be very similar, and the importance of their relationship to each other may be stressed. 

A further close link between cis-dehydratase activity and aromatic and fatty acid synthetases became apparent when it was observed that chloro-plasts, isolated from etiolated barley leaves that had been partially greened, formed 6-methylsalicylate at the expense of fatty acids (Kannangara et al., 1971). The requirement for NADPH would be met in this system from endogenous sources. Mature chloroplasts and etioplasts do not possess this capacity for phenol production and presumably lack the ability to make the synthetase responsible. 

The polyketide synthases and peptide synthetases catalyze an unusually large number of reactions. 6-Methylsalicylic acid synthase, which produces 6-methylsalicylic acid from acetyl Co A, malonyl Co A and NADPH, for instance, carries out a total of thirteen reactions. The peptide antibiotic synthetases activate each of the amino acids involved in the formation of the peptides by a two step mechanism at a specific peripheral domain of the enzyme protein and catalyze also linkage of the activated amino acids by sequential transpeptidation. In the case of gramicidin S synthetase for example, there are twenty-one different reactions carried out by two multifunctional enzyme proteins. 

Vogel, G., and F. Lynen 6-Methylsalicylic Acid Synthetase. Methods in Enzymo-logy, Vol. 43, Antibiotics Edit. Hash, J. H., p. 520. Academic Press. 1975. 

Exciting progress - has been made in the enzymology of systems which elaborate phenolic compounds by the acetate-poly maionate pathway. The purification and properties of 6-MSA (6-methylsalicylic acid) (4) synthetase were reported in 1970 and recent work has provided further information on this enzymic system. Thus it has been reported that an ammonium sulphate protein fraction from P. patulum catalyses the synthesis of 6-MSA, TAL (triacetic acid lactone) (2), and fatty acids in the presence of malonyl-coenzyme A and NADPH. The derivation of 6-MSA from one acetate and three maionate... 

Salicylic Acid.—It has recently been demonstrated (see last year s Report ) that whereas 6-methylsalicylic acid in both micro-organisms and higher plants is acetate-derived, salicylic add itself is formed in both groups of organisms by the shikimic add pathway. Further confirmation of this has come from Marshall and Ratledge, who have studied the enzymology of biosynthesis of salicylic acid in Mycobacterium smegmatis. These authors isolated the enzyme salicylate synthetase, which catalyses the last step (see Scheme 1) in synthesis of salicylic acid (1) from isochorismic add (2), formed in turn from chorismic (3) and shikimic adds (4). The enzyme has no cofactor requirements and converts (2) directly into (1). No evidence could be obtained for the presence in bacterial cultures of the possible intermediate 2,3-dihydroxy-2,3-dihydrobenzoic add. 

Analogy between 6-Methylsalicylic Acid Synthetase and Yeast Fatty Acid Synthetase... 

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