Shikimate pathway evidence

The mutants that grew in the presence of shikimic acid evidently had the biosynthetic pathway blocked... 

MK biosynthesis by the OSB pathway has been elucidated on the basis of isotopic tracer experiments, isolation of mutants blocked in the various steps, isolation and identification of intermediates accumulated by the mutants, and by enzyme assays. Early isotopic tracer experiments with various bacteria established that methionine and prenyl PPi contribute to the methyl and prenyl substituents of the naphthoquinone. The early isotopic tracer studies and other work have been reviewed by Bentley and Meganathan. " In 1964, Cox and Gibson observed that [G- " C] shikimate was incorporated into both MK and ubiquinone by E. coli, thus providing the first evidence for the involvement of the shikimate pathway." Chemical degradation of the labeled isolated menaquinone (MK-8) showed that essentially all of the radioactivity was retained in the phthalic anhydride. It was concluded that the benzene ring of the naphthoquinone (sic) portion of vitamin K2 arises from shikimate in E. coli The authors further suggested that shikimate was first converted to chorismate before incorporation into MK. A more complete chemical degradation of the MK derived from... 

Phytoalexins can be elicited in plants by a variety of agents, including viruses, microorganisms and nematodes [292, 293]. When resistant plants are infected by nematodes, phytoalexins with antinematodal activity can be produced [294]. Some coumestans have been implicated in the resistance of plants to nematodes. For example, when the roots of the resistant lima bean (Phaseolus lunatus) were inoculated with Pratylensus scribneri, coumestrol (181) accumulated at the site of nematode attack. In vitro, coumestrol (5 ig/ml) inhibited the motility of the nematode [294]. Correlative evidence for a functional role of related compounds in resistance towards nematodes has been obtained [295-297]. In particular, nematode attack on the roots elicits the transcription of genes encoding several enzymes of the shikimate pathway that leads to phytoalexin production. Induction of enzyme activity results from transcriptional activation of the genes leading to increased levels of translatable mRNA [298]. 

Rifamycins.—Contained within the rifamycin skeleton, for example that of rifamycin S (169), is a C7-N unit (heavy bonding) which has been deduced to arise from an intermediate of the shikimic acid pathway. Fresh evidence arising from a study using mutants of Nocardia meditenanei confirms this view, and it is apparent that divergence from the shikimate pathway to rifamycin synthesis occurs between sedoheptulose-7-phosphate and shikimic acid itself. 

There is growing evidence that the shikimate pathway enzymes exist in the chloroplast as well as in the cytoplasm. Shikimate dehydrogenase isoen-... 

Amplification of the oxidation-reduction potential is not the only cause for diversity of secondary metabolites in angiosperms. In order to understand the phenomenon more fully it is necessary to remember also the best known of the evolutionary trends the gradual substitution of woody forms by herbaceous ones that operate in several lineages. Reduced utilization for the production of lignins and condensed tannins should cause initially a surplus of cinnamate, raw material for the biosynthesis of allelochemicals of primitive angiosperms. The continuing decrease in the importance of the shikimate pathway and the connection between primary, and secondary metabolism requires that the transition from the woody to the herbaceous habit be accompanied in the long run by a curtailment of shikimate precursors for the biosynthesis of allelochemicals. Let us look now into the evidence for these postulates. 

Evidence for the operation of the shikimate pathway in higher plants has been obtained mainly from tracer experiments and from a limited range of enzyme studies. These have shown that not only do the pathways of biosynthesis of L-phenylalanine, L-tyrosine and L-tryptophan involve the same intermediates as in bacteria and fungi Figures 1.2, 1.13, 1.14), but that higher plants can also convert the aromatic amino acids into a plethora of characteristic natural products or secondary metabolites ... 

The evidence that (- )-shikimic acid plays a central role in aromatic biosynthesis was obtained by Davis with a variety of nutritionally deficient mutants of Escherichia coli. In one group of mutants with a multiple requirement for L-tyrosine, L-phenylalanine, L-tryptophan and p-aminobenzoic acid and a partial requirement for p-hydroxybenzoic acid, (—)-shikimic acid substituted for all the aromatic compounds. The quintuple requirement for aromatic compounds which these mutants displayed arises from the fact that, besides furnishing a metabolic route to the three aromatic a-amino acids, the shikimate pathway also provides in micro-organisms a means of synthesis of other essential metabolites, and in particular, the various isoprenoid quinones involved in electron transport and the folic acid group of co-enzymes. The biosynthesis of both of these groups of compounds is discussed below. In addition the biosynthesis of a range of structurally diverse metabolites, which are derived from intermediates and occasionally end-products of the pathway, is outlined. These metabolites are restricted to certain types of organism and their function, if any, is in the majority of cases obscure. 

The carbon flow from 3-phosphoglycerate, phosphoenolpyruvate, pyruvate and acetyl-CoA. Even if the synthesis of aromatic amino acids by shikimate pathway /28,29,30,31/ and also prenyl-PP synthesis via mevalonate /32,33,34/ has been established in chloroplasts by identification of respective plastidic enzymes, it is still a matter of discussion from where PEP origins to supply DAHP synthesis of the shikimate pathway and from where pyruvate is delivered to supply the plastidic pyruvate dehydrogenase complex (for isolation see Treede and Heise, this Conference). Because phosphoglycerate mutase (PGM) to form 2-PGA from 3-PGA could not be detected in chloroplasts /35/ and acetyl-CoA is preferably synthesized from added acetate by the actetyl-CoA synthetase /36/, particularly in spinach chloroplasts, it was argued that chloroplasts are dependent on import of these substrates from the external site. Evidence for PEP formation from 3-PGA within the chloroplast could be obtained by three different approaches (D. Schulze-Siebert, A. Heintze and G. Schultz, in preparation D. Schulze-Siebert and G. Schultz, in preparation, for plastidic isoenzyme of PGM in Ricinus see /37/ and in Brassica /38/). 

The manner of formation of the aromatic ring has been studied mainly in microorganisms using the techniques of biochemical genetics. A determined search provided mutants in which the chain of synthetic steps was interrupted and which required— in contrast to the wild strains—a supplement of aromatic compounds (phenylalanine, tyrosine cf. also Chapt. VII-6). One mutant of E. coli was found to need five different aromatic acids tyrosine, phenylalanine, tryptophan, p-aminobenzoic acid, and p-hydroxybenzoic acid. AH the requirements of this particular strain, however, were met by a single substance, shikimic add. Evidently aU five synthetic pathways begin with this common precursor. 

Theoretically, many of the above discrepancies could be settled by experiments with carboxyl-labeled shikimic acid because this functional group would be lost in the formation of phenylalanine, but retained in the case of a direct conversion to gallic acid. Only ambiguous evidence was obtained, however, from such efforts (10), and it was concluded that at least two pathways for gallic acid biosynthesis must exist (14), with the preferential route depending on leaf age and plant species investigated (15,16). 

In contrast to the rutelines, the melolonthine scarabs generally use terpenoid-and amino acid-derived pheromones (reviewed in Leal, 1999). For example, the female large black chafer, Holotrichia parallela Motschulsky, produces methyl (2.S, 3. Sj - 2 - am ino-3-methy lpcn tanoatc (L-isoleucine methyl ester) as an amino acid-derived sex pheromone (Leal et al., 1992 Leal, 1997). There is no direct evidence that the chafer beetles or any other Coleoptera use the shikimic acid pathway for de novo pheromone biosynthesis, but some scarabs and scolytids (see section 6.6.4.2) may convert amino acids such as tyrosine, phenylalanine, or tryptophan to aromatic pheromone components (Leal, 1997,1999). In another melolonthine species, the female grass grab beetle, Costelytra zealandica (White), the phenol sex pheromone is produced by symbiotic bacteria (Henzell and Lowe, 1970 Hoyt et al. 1971). 

The evidence then is that, for rifamycin and other ansamycins, biosynthesis diverts at a so-far unidentified (but early) compound in the shikimic acid pathway to give 3-amino-5-hydroxybenzoic acid (91) (as its CoA ester). This compound then yields, on the one hand, the mitomycins [e.g. porfiromycin (88)1 and, on the other, the CoA ester of P8/1-OG (92), which then affords diverse metabolites such as rifamycin B (87) and actamycin (86) (cf. ref. 83 for a detailed scheme). 

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