Elucidation of Biosynthetic Pathways

Particularly important to the pathways of modular synthases is the incorporation of novel precursors, including nonproteinogenic amino acids in NRP systems [17] and unique CoA thioesters in PK and fatty acid synthases [18]. These building blocks expand the primary metabolism and offer practically unlimited variability applied to natural products. Noteworthy within this context is the contiguous placement of biosynthetic genes for novel precursors within the biosynthetic gene cluster in prokaryotes. Such placement has allowed relatively facile elucidation of biosynthetic pathways and rapid discovery of novel enzyme mechanisms to create such unique building blocks. These new pathways offer a continued expansion of the enzymatic toolbox available for chemical catalysis. 

Schmidt H-L, Werner RA, Eisenreich W (2003) Systematics of H patterns in natural compounds and its importance for the elucidation of biosynthetic pathways. Phytochem Rev 2 61 Schmidt H-L, Werner RA, Rofimann A (2001) Pattern and biosynthesis of natural plant products. Phytochemistry 58 9... 

The application of 13C NMR spectroscopy has become one of the most informative standard methods for the elucidation of biosynthetic pathways. This is demonstrated by numerous examples reported in the literature [998 1003] and by some selected examples summarized in this section. By adding 13C-enriched precursors to culture media, specifically labeled biosynthetic materials can be isolated. 13C-enriched positions are easy to identify as they cause larger signal intensities in the 13C NMR spectrum. In most cases an enrichment of 0.5% 13C above natural abundance is sufficient to detect the labeled site. 

Aglycones obtained from one macrolide have been bioconverted by microorganisms producing either the same or a different macrolide such studies have been important for production of new compounds and elucidation of biosynthetic pathways. 

Some of the most interesting applications of organic structural theory to the elucidation of biosynthetic pathways were stimulated by efforts to formulate mechanisms for the biosynthesis of alkaloids. Conversely, consideration of implied biogenetic relations have occasionally helped structural determination. An important aspect of theories concerning alkaloid biosynthesis has been the assumed role of the aromatic amino acids in their formation. Only limited experimental evidence is available in this area. The incorporation of tyrosine- 8-C into morphine has been shown to be in accordance with a theory for its formation from 3,4-dihydroxyphenyl-alanine plus 3,4-dihydroxyphenylacetaldehyde. A stimulating theory of the biosynthesis of indole alkaloids, based on a condensation between trypt-amine and a rearrangement product of prephenic acid, has recently been published. The unique stereochemistry of C15 of these alkaloids had an important part in the formulation of the theory. Experimental proof of this theory would be valuable for several areas of alkaloid chemistry and biosynthesis. 

The existence of a second pathway that affords IPP and DMAPP was discovered in the 1990s. The details of that non-mevalonate pathway were then established in rapid sequence by the combination of isotope studies, comparative genomics, and enzymology (52). The delayed discovery of the nonmevalonate pathway can serve as paradigm for pitfalls in the elucidation of biosynthetic pathways. 

Equally important as NMR spectroscopy is the development of mass spectrometry where advances have been even more spectacular, partly due to its far greater sensitivity. A broad introduction is provided by Hocart (Chapter 9.10), who describes the range of available spectrometers and techniques. In the next chapter (Chapter 9.11), Dorrestein and his coauthors describe applications to the elucidation of biosynthetic pathways, focusing on nonribosomal peptides and polyketides, while Das provides a detailed account of the structure determination of proteins and peptides in Chapter 9.12. In yet another illustration of the power of mass spectrometry to analyze biomacromolecules, Li and Altman extend the technique to a study of bacterial polysaccharides (Chapter 9.13). 

For the past 20 years, there has been a growing interest in applying the in vitro culture of plant cells, tissues and organs to the study of medicinal plants. Main topics include the development of culture for the large-scale production of valuable chemicals, the discovery of new biologically active metabolites, the selection of plants showing favourable characteristics of productivity, the elucidation of biosynthetic pathways with isolation of... 

Until such time when the biosynthetic pathways of the complex indole alkaloids have been completely defined their full taxonomic value cannot be appraised. However, a number of recent reviews (7, 8) indicate that chemotaxonomic considerations in this area have been aided by the rapid progress made in the chemistry and biosynthesis of the various indole alkaloids. Furthermore, the latter two disciplines have benefited from the former in the search for new alkaloids and in the elucidation of biosynthetic pathways. In the future it is expected that many advances in indole alkaloid chemistry will often arise as a result of the convergence of biochemical and chemical research efforts (9). 

All of the cyclic imine toxins (except spiro-prorocentrimine) may be structurally classihed as macrocyclic polyethers. Although the elucidation of biosynthetic pathways for polyether dino-flagellates toxins is limited to the ladder frame polyether brevetoxins [40], the linear polyether dinophysistoxins DTX4 and DTX5 [11,41] and the macrocyclic imine des-methyl spirolide C... 

Some of the most fascinating work described in this section deals with the elucidation of biosynthetic pathways by NMR spectroscopy. Brown and... 

The elucidation of biosynthetic pathways uses nowadaj isotopicaUy labelled precursors. Labelling experiments are frequently carried out with the radioactive C-isotope. Once the radioactive metabolites are identified, the precise position of the label can be determined by degradation reactions. For C-labelled compounds, there is also the possibility, not only to identify the metabolites with the aid of modern NMR spectroscopy, but also to directly resolve their structure and labelling pattern. 

Richard Heys received his Ph.D. in organic chemistry from Stanford University in 1976 and conducted postdoctoral research in the chemistry department at Yale both involved the synthesis of radiolabeled compounds and their use in elucidation of biosynthetic pathways. His subsequent 29-year career in organic radiochemical synthesis both in the laboratory and as a manager took him to the Radiochemistry Department of Midwest Research Institute (now part of Aptuit, Inc.), Smith Kline French Laboratories/SmithKline Beecham Pharmaceuticals (now GlaxoSmithKline) and AstraZeneca Pharmaceuticals. Author or coauthor of over 85 publications, 8 patents and a previous conference proceedings volume in the field, organizer of an international symposium on the synthesis of isotopicaUy labeled compound and holder of leadership positions (including president and CFO) in the International Isotope Society for 9 years, he is retired and lives in northwestern Connecticut. 

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