Tracing Biosynthetic Pathways

In studying biosynthetic pathways, we have to identify (a) the ultimate source in primary metabolism from which the compound of interest derives (for example, fatty acid, polyketide, or others in the following chapters), and (b) the intermediates through which a final product is formed. With so much accumulated knowledge, and with only a few pathways used by nature, the first task of finding the ultimate source is usually not at all difficult. The second objective may be very difficult and subject to all sorts of pitfalls and false clues.

The usual method of study is to suggest a possible precursor and to feed it to the biosynthesizing system. The precursor has to be labelled in some way to trace it through the sequence of reactions, and that is usually by some isotopic element. It may be a radio-active isotope, such as 3H, 14C, 32P or 35S, that can be followed by its radiation; or it can be a stable heavy isotope, such as 2H, 13C, I5N, or lsO, that can be traced by mass spectrometry or nuclear magnetic resonance (NMR) spectroscopy (Table 5.1). Another possible way is to use mutant strains of an organism that lack the enzymes to complete a particular synthesis, or to add a specific enzyme inhibitor, so that intermediates accumulate and can be identified. A mutant strain of yeast was important in discovering mevalonic acid and its place in terpene biosynthesis (Chapter 6) and a number of mutants of the bacterium Escherichia coli helped to understand the shikimic acid pathway (Chapter 8).

In the simplest kind of experiment, a labelled potential precursor may be fed to, or injected into, an animal or added to an in vitro system and labelling sought in the product of interest. Because many compounds can be broken down to acetate and then re-built into other compounds, this kind of experiment may not be conclusive. This was the case with the labelling study of dendrolasin (Figure 6.17).

Table 5.1 Some isotopes used in biosynthesis studies and how they are detected

Isotope

Natural abundance

Detection method

Nuclear spin

>H

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