Mutants metabolism

Genetic disposition, either by interaction of the drug with a mutant target or by the lack of certain (or mutant) metabolic enzyme (e.g., the inability of about 1—3% of the Caucasian population to metabolize S-warfarin, due to a CYP2C9 deficiency). 

As an alternative to the use of mutants, metabolic inhibitors may be used to interrupt metabolic pathways even transient intermediates may then be accumulated and provide evidence for the details of the consecutive steps. A wide range of compounds has been used in investigations on electron transport pathways and bioenergetics, but these lie beyond the scope of this account. Examples that have been used in metabolic studies with bacteria include molybdate as an inhibitor of anaerobic sulfate reduction, methyl fluoride and difluoromethane (Miller et al. 1998) as inhibitors of the aerobic... 

Tlie analysis of the intracellular metabolites, the metabolome, by means of comparison of mutants metabolic profiles (comparative metabolomics) aids in understanding the networks of proteins and the presence of silent genes. 

Hundreds of metabohc reac tions take place simultaneously in cells. There are branched and parallel pathways, and a single biochemical may participate in sever distinct reactions. Through mass action, concentration changes caused by one reac tion may effect the kinetics and equilibrium concentrations of another. In order to prevent accumulation of too much of a biochemical, the product or an intermediate in the pathway may slow the production of an enzyme or may inhibit the ac tivation of enzymes regulating the pathway. This is termed feedback control and is shown in Fig. 24-1. More complicated examples are known where two biochemicals ac t in concert to inhibit an enzyme. As accumulation of excessive amounts of a certain biochemical may be the key to economic success, creating mutant cultures with defective metabolic controls has great value to the produc tion of a given produc t. 

The same is true of the thiazole acid 40. Although discovered as a growth factor, it is unable to sustain the growth of a thiazole-deficient mutant of E. coli in a liquid medium. It does not decarboxylate in water solution at pH 7. Phosphate 41 (Scheme 17) is also biologically inactive. In any case, if there is only one metabolic route to the thiazole of thiamine, the very structures of 39 and 40 show that they cannot both be intermediates. 

Metabolic pathways containing dioxygenases in wild-type strains are usually related to detoxification processes upon conversion of aromatic xenobiotics to phenols and catechols, which are more readily excreted. Within such pathways, the intermediate chiral cis-diol is rearomatized by a dihydrodiol-dehydrogenase. While this mild route to catechols is also exploited synthetically [221], the chirality is lost. In the context of asymmetric synthesis, such further biotransformations have to be prevented, which was initially realized by using mutant strains deficient in enzymes responsible for the rearomatization. Today, several dioxygenases with complementary substrate profiles are available, as outlined in Table 9.6. Considering the delicate architecture of these enzyme complexes, recombinant whole-cell-mediated biotransformations are the only option for such conversions. E. coli is preferably used as host and fermentation protocols have been optimized [222,223]. 

Resistance to DDT has been developed in many insect species. Although there are some cases of metabolic resistance (e.g., strains high in DDT dehydrochlorinase activity), particular interest has been focused on kdr and super kdr mechanisms based upon aberrant forms of the sodium channel—the principal target for DDT. There are many examples of insects developing resistance to dieldrin. The best-known mechanism is the production of mutant forms of the target site (GABA receptor), which are insensitive to the insecticide. 

Fig. 25.8 (a) Normal metabolism, in which phenylalanine is converted by phenylalanine 4-mono-oxygenase to tyrosine, (b) Phenylketonuria, in which there is a transamination reaction between phenylalanine and a-ketoglutaric acid. Phenylalanine 4-mono-oxygenase is absent in about 1 in every 10000 human beings because of a recessive mutant gene. 

Generation of mutants is also a starting point in optimization experiments, and now is the time for metabolic engineering of the astaxanthin biosynthetic pathway. Researchers should be able to manage carbon fluxes within the cells and resolve competitions between enzymes such as phytoene desaturase and lycopene cyclase. 

Although uptake and accumulation of most amino acids from the external medium seems to be irreversible, amino acids are excreted into the medium whenever they are overproduced above a given threshold by yeast cells [6], This can occur under a number of specific conditions, namely in mutants with impaired regulation of amino acid biosynthesis, or in the presence of mutations preventing substrate catabolism, or when growth occurs in the presence of metabolic intermediates. It can even occur when growth is arrested under conditions where amino acid synthesis can continue. 

In contrast, less effort has apparently been directed to the transport of xenobiotics, and there is an intrinsic difficulty that in contrast to organisms that utilize carbohydrates or amino acids, suitable mutants defective in the metabolism of the substrate may not be available. This limitation makes it impossible to determine directly whether active transport is involved. Although the genes encoding permeases have been described quite frequently, details of their mechanisms have been less well documented ... 

Harder PA, DP O Keefe, JA Romesser, KJ Leto, CA Omer (1991) Isolation and characterization of Strepto-myces griseolus deletion mutants affected in cytochrome P-450-mediated herbicide metabolism. Mol Gen Genet 227 238-244. 

Reiner AM, GD Hegeman (1971) Metabolism of benzoic acid by bacteria. Accumulation of (-)-3,5-cyclo-hexadiene-l,2-diol-l-carboxylic acid by a mutant strain of Alcaligenes eutrophus. Biochemistry 10 2530-2536. 

A mutant strain of Alcaligenes eutrophus was used (Reiner and Hegeman 1971) to demonstrate that the initial step in the metabolism of 4-fluorobenzoate involves 1 2 dioxygenation to 4-fluoro-1,2-dihydrodihydroxybenzene-l-carboxylate. 4-Fluorobenzoate may be used for growth by several bacteria, and metabolites produced by a strain of Pseudomonas sp., which was also able to degrade... 

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