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 H, 0, or that can be followed by its radiation or it can be a stable heavy isotope, such as H, C, N, or 0, 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). 

Isotope Natural abundance Detection method Nuclear spin 

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Figure 11-4 Cascades of phosphorylation and dephosphorylation reactions involved in the control of the metabolism of glycogen. Heavy arrows show pathways by which glucosyl emits of glycogen are converted into free glucose or enter the glycolytic pathway. Green arrows trace the corresponding biosynthetic pathways. Gray arrows (— ) trace the...

Figure 17-11 Some major biosynthetic pathways. Some key intermediates are enclosed in boxes and the 20 common amino acids of proteins are encircled. Key intermediates for each family are in shaded boxes or elipses. Green lines trace the reactions of the glyoxylate pathway and of glucogenesis.

Stereospecific 2,3-epoxidation of squalene. followed by a non-concerted carbocationic cyclization and a seiies of carbocationic rearrangements, forms lanosterol (26) in the first steps dedicated solely toward steroid synthesis. Cholesterol is the principal starting material for steroid hormone biosynthesis ill animals. The cholesterol biosynthetic pathway is composed of at least 30 enzymatic reactions. Lanosterol and squalene appear to he normal constituents, in trace amounis. in tissues that are actively synthesizing cholesterol,... 

The foregoing discussion has attempted to trace the ways in which cholesterol, derived from plasma lipoproteins, is converted into the various steroid hormones and how these are secreted back into the blood. Of necessity, many details have had to be omitted but it is hoped that this up-date has shown the complexities of steroid biosynthetic pathways and that earlier classical ideas have had to be modified as greater knowledge of intermediates, isoenzymes and multiple forms of cyt P-450s has become available. Perspectives for future studies are indeed exciting. 

The first stable product of carbon fixation by the enzyme, ribulose bisphosphate carboxylase (Rubisco), is glyceraldehyde 3-phosphate, a 3-C sugar. This 3-C sugar is fed into biosynthetic pathways and forms the basis for all organic compounds produced by photosynthetic organisms. Fixed carbon and major and trace elements... 

Moreover, the treatment of methylvelutinal (7.13) with lithium diisopropylamide afforded, by a P-elimination of the epoxyde, the corresponding 7,13-en-8a-ol derivative 7.9 (36). This product can be easily hydrolysed to isovelleral (6.1) in a THF/H2O mixture containing traces of acid or on prolonged contact with silica gel (36). This conversion may support the biosynthetic pathway proposed for the transformation of velutinals to isovelleral (6.1) in injured mushrooms (36,46). 

In 1995, Bonnarme et al. [110] used the analytical techniques that combine isotopic tracing, nuclear magnetic resonance spectroscopy, and mass spectroscopy to compare the enzyme systems of intact cells of high- and low-producing strains of A. terreus. Results show that itaconate formation requires de novo protein synthesis. During acid formation, TCA cycle intermediates increase in both strains. Furthermore, data showed that both the BMP pathway and the TCA cycle are involved in itaconate biosynthesis. Based on the biosynthetic pathway (Fig. 15), one itaconate molecule is produced from one hexose molecule with a theoretical weight yield of 72.2%. The actual yield should be lower due to the loss of carbon to biomass accumulation and cell maintenance. 

The commonest enzymatic deficiency is a relatively severe or total absence of steroid 21-hydroxylase, but minor or severe deficiencies in the 11 -hydroxylase, and in A -3j8-hydroxysteroid dehydrogenase also occur. As with any metabolic defect of this sort, products of the reactions prior to the deficient step reach abnormally high steady-state concentrations, and their conversion by side branches of the main biosynthetic pathway to usually minor or trace metabolites may become spectacularly prominent. 

Mevalonic acid (MVA) (69) is a very important biosynthetic intermediate used by us to make steroids and by plants to make terpenes. Tracing these biosynthetic pathways required radioactively labelled MVA and J. W. Cornforth has published many such syntheses. MVA has two 1,3-dicarbonyl relationships (70), one of which can be disconnected to (71) and (72). If the OH and CO2H groups are both protected as esters, we can use the Reformatsky method to activate (72) and finally remove both protecting groups in one step. 

The generation from a producing organism of mutant strains that are blocked or altered in the biosynthetic pathway can lead to the isolation of related metabolites that could not otherwise be obtained. These metabolites may be intermediates from the blocked pathway that would normally be transient and detectable only in trace amounts, if at all, or they may represent shunt metabolites, whereby the intermediates have gone down a different biosynthetic route, resulting in novel compounds. The products of such mutants may be of interest in their own right, or they may be of interest in biosynthetic studies, biotransformation experiments or as starting points for precursor-directed biosynthesis. 

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