Isoleucine, synthesis

It was snbseqnently discovered that the first enzyme in the pathway for isoleucine synthesis, which is threonine deaminase, was inhibited by isoleucine in an extract of E. coli. No other amino acid caused inhibition of the enzyme. Threonine deaminase is, in fact, the rate-limiting enzyme in the pathway for isoleucine synthesis, so that this was interpreted as a feedback control mechanism (Fignre 3.13(a)). Similarly it was shown that the hrst enzyme in the pathway for cytidine triphosphate synthesis, which is aspartate transcarbamoylase, was inhibited by cytidine triphosphate (Fignre 3.13(b)). Since the chemical structures of isoleucine and threonine, or cytidine triphosphate and aspartate, are completely different, the qnestion arose, how does isolencine or cytidine triphosphate inhibit its respective enzyme The answer was provided in 1963, by Monod, Changenx Jacob. 

The most responsive regulation of amino acid synthesis takes place through feedback inhibition of the first reaction in a sequence by the end product of the pathway. This first reaction is usually irreversible and catalyzed by an allosteric enzyme. As an example, Figure 22-21 shows the allosteric regulation of isoleucine synthesis from threonine (detailed in Fig. 22-15). The end product, isoleucine, is an allosteric inhibitor of the first reaction in the sequence. In bacteria, such allosteric modulation of amino acid synthesis occurs as a minute-to-minute response. 

The present studies confirm the earlier studies indicating the relatively great biosynthetic abilities of the methane bacteria and suggest that much of the cellular carbon compounds are probably synthesized from acetate and carbon dioxide. In view of the carbon dioxide and acetate requirements and the reductive carboxylation reactions shown to be involved in isoleucine synthesis in M. ruminantium (26) and the probability of similar carboxylation reactions in biosynthesis of isoleucine, alanine, and other amino acids in MOH, suggested by the studies on M. omelianskii (34), the operation of the pyruvate synthase reaction and some other reactions of the reductive carboxylic acid cycle (35, 36) as major pathways of biosynthesis of cellular materials in these bacteria is an attractive hypothesis. 

The syntheses of valine, leucine, and isoleucine from pyruvate are illustrated in Figure 14.9. Valine and isoleucine are synthesized in parallel pathways with the same four enzymes. Valine synthesis begins with the condensation of pyruvate with hydroxyethyl-TPP (a decarboxylation product of a pyruvate-thiamine pyrophosphate intermediate) catalyzed by acetohydroxy acid synthase. The a-acetolactate product is then reduced to form a,/3-dihydroxyisovalerate followed by a dehydration to a-ketoisovalerate. Valine is produced in a subsequent transamination reaction. (a-Ketoisovalerate is also a precursor of leucine.) Isoleucine synthesis also involves hydroxyethyl-TPP, which condenses with a-ketobutyrate to form a-aceto-a-hydroxybutyrate. (a-Ketobutyrate is derived from L-threonine in a deamination reaction catalyzed by threonine deaminase.) a,/3-Dihydroxy-/3-methylvalerate, the reduced product of a-aceto-a-hydroxybutyrate, subsequently loses an HzO molecule, thus forming a-keto-/kmethylvalerate. Isoleucine is then produced during a transamination reaction. In the first step of leucine biosynthesis from a-ketoisovalerate, acetyl-CoA donates a two-carbon unit. Leucine is formed after isomerization, reduction, and transamination. 

The valine and isoleucine biosynthetic pathways share four enzymes. Isoleucine synthesis begins with the reaction of a-ketobutyrate (a derivative of threonine) with pyruvate. In valine synthesis the condensation of two pyruvate molecules is the first step. Leucine is produced by a series of reactions that begin with a-ketoisovalerate, an intermediate in valine synthesis. 

Fig. 5-32 Metabolic control network for isoleucine synthesis by Rh. spheroides.

In an analogous manner, a carboxyl group may also be transferred to propionyl-CoA (for the biosynthesis of branched or odd-numbered fatty acids, isoleucine synthesis, or cholesterol metabolism) or to 3-methylcrotonyl-CoA (a degradation product of leucine after addition of water, there results hydroxymethyl-glutaryl-CoA, the precursor of mevalonic acid cf. section 7.1.2). [117] Oxaloacetic acid is derived from pyruvate, which is of central importance for gluco-neogenesis. [108] In addition, biotin participates also in the transfer of carboxylic acid functions. In prokaryotes, biotin functions as a cofactor for decarboxylases (Tab. 7.6). 

In the next two rounds of selection, an additional introducing an ilvl deletion was introduced, to reduce enzyme affinity towards 2-ketobytirate and lower isoleucine synthesis. This was another eliminating criterion for CimA expression, since strain growth is determined by the ability of CimA to provide precursors for L-isoleucine synthesis (Atsumi and Liao 2008b). 

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