Keto-acid pathway

Another method for alcohol production is through the keto-acid pathways [62] (Figure 5.2). Multiple keto-acids exist in E. coli metabolism as intermediates to various products, particularly amino acids. The most common of these is the 3-carbon keto-acid pyruvate. Keto-acids can be decarboxylated to aldehydes, which in turn can be reduced to alcohols. In fact, decarboxylation of pyruvate to acetaldehyde followed by reduction to alcohol is the basis for bioethanol production. Extending this concept, any keto-acid can be converted to the corresponding... 

Figure 15.4 Isobutanol and 2-methyl butanol (2MB) production via keto acid pathway in cyanobacteria. Gene/protein symbols are dmA, citramalate synthase leuCD, isopropylmalate isomerase leuB, 3-isopropylmalate dehydrogenase AHAS, acetohydroxyacid synthase ilvC, acetohydroxy acid isomeroreductase ilvD, dihydroxy acid dehydratase kivd, ketoisovalerate decarboxylase yqhD, alcohol dehydrogenase.

Shen CR, Liao JC. (2008). Metabolic engineering of Escherichia coli for 1-butanol and 1-propanol production via the keto-acid pathways. Metab Eng, 10, 312—320. 

Butanol has been synthesized in various organisms through different pathways. The CoA-dependent pathway from Clostridium and its variations (Figure 19.1) are the best studied [65]. This pathway is chemically similar to the reversal of P-oxidization [66, 67]. The intrinsic iteration nature of reverse p-oxidization also enables the biosynthesis of longer chain alcohols, for example, -hexanol and n-octonol [66]. The citramalate and threonine pathways fall into the same broad group as the keto-acid pathway (Figure 19.2), which is more commonly utihzed for isobutanol production. 

Other Products from the Keto-Acid Pathway... 

A rather limited collection of simple precursor molecules is sufficient to provide for the biosynthesis of virtually any cellular constituent, be it protein, nucleic acid, lipid, or polysaccharide. All of these substances are constructed from appropriate building blocks via the pathways of anabolism. In turn, the building blocks (amino acids, nucleotides, sugars, and fatty acids) can be generated from metabolites in the cell. For example, amino acids can be formed by amination of the corresponding a-keto acid carbon skeletons, and pyruvate can be converted to hexoses for polysaccharide biosynthesis. 

Pyruvate kinase possesses allosteric sites for numerous effectors. It is activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine. (Note that alanine is the a-amino acid counterpart of the a-keto acid, pyruvate.) Furthermore, liver pyruvate kinase is regulated by covalent modification. Flormones such as glucagon activate a cAMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the enzyme. The phos-phorylated form of pyruvate kinase is more strongly inhibited by ATP and alanine and has a higher for PEP, so that, in the presence of physiological levels of PEP, the enzyme is inactive. Then PEP is used as a substrate for glucose synthesis in the pathway (to be described in Chapter 23), instead... 

TPP-dependent enzymes are involved in oxidative decarboxylation of a-keto acids, making them available for energy metabolism. Transketolase is involved in the formation of NADPH and pentose in the pentose phosphate pathway. This reaction is important for several other synthetic pathways. It is furthermore assumed that the above-mentioned enzymes are involved in the function of neurotransmitters and nerve conduction, though the exact mechanisms remain unclear. 

In intact cell systems or vivo, the primary products of a-hydroxylation, 22. have not been detected. The principal urinary metabolites of NNN resulting from a-hydroxylation are keto acid 21 from 2 -hydroxyl at ion and hydroxy acid 21 from 5 -hydroxylation. Trace amounts of 7 y 21> H ve also been detected as urinary metabolites (34). The interrelationships of these metabolites as shown in Figure 2 have been confirmed by administration of each metabolite to F-344 rats (37). The other metabolites which are routinely observed in the urine are NNN-1-N-oxide U1 and 5-(3-pyridyl)-2-pyrrolidinone [norcotinine, ]. The p-hydroxy derivatives 2. 1 were also detected in the urine of NNN treated rats, but at less than 0.1% of the dose (36). An HPLC trace of the urinary metabolites of NNN is shown in Figure 3. Urine is the major route of excretion (80-90% of the dose) of NNN and its metabolites in the F-344 rat in contrast to NPYR which appears primarily as CO2 (70%) after a dose of 16 mg/kg (17). This is because the major urinary metabolite of NNN, hydroxy acid 21> fs not metabolized further, in contrast to 4-hy-droxybutyric acid [2, Figure 1] which is converted to CO2. In addition, a significant portion of NNN is excreted as NNN-l-N-oxide U ], a pathway not open to NPYR. 

Phenylglycines are important components of the vancomycin/teicoplanin antibiotics, and the conforma-tionally restricted amino acids contribute to the unique architecture and biological function of these clinically important NRPs. 4-Hydroxyphenylglycine is produced from L-tyrosine in a pathway that involves three enzymes. In the key step, a nonheme iron oxidase catalyzes the oxidative decarboxylation of the a-keto acid derivative of L-tyrosine resulting in loss of carbon dioxide and generation of the phenylglycine carbon framework. 

In enzymic decarboxylations the mechanistic pathway seems to involve Schiff base formation between an —NH2 from a lysine residue and a C=0 of the keto acid.52 Likewise, with small-molecule primary amines, catalysis of decarboxylation of /3-ketoacids53-58 has been ascribed to a Schiff base intermediate. The overall reaction for oxalacetate is... 

FIGURE 18-28 Catabolic pathways for the three branched-chain amino acids valine, isoleucine, and leucine. The three pathways, which occur in extrahepatic tissues, share the first two enzymes, as shown here. The branched-chain -keto acid dehydrogenase complex... 

Alanine, aspartate, and glutamate are synthesized by transfer of an amino group to the a-keto acids pyruvate, oxaloacetate, and a-keto-glutarate, respectively. These transamination reactions (Figure 20.12, and see p. 248) are the most direct of the biosynthetic pathways. Glutamate is unusual in that it can also be synthesized by the reverse of oxidative deamination, catalyzed by glutamate dehydrogenase (see p. 249). 

TPP-dependent enzymes catalyze either simple decarboxylation of a-keto acids to yield aldehydes (i.e. replacement of C02 with H+), or oxidative decarboxylation to yield acids or thioesters. The latter type of reaction requires a redox coenzyme as well (see below). The best known example of the former non-oxidative type of decarboxylation is the pyruvate decarboxylase-mediated conversion of pyruvate to acetaldehyde and C02. The accepted pathway for this reaction is shown in Scheme 10 (69MI11002, B-70MI11003, B-77MI11001>. 

However, now it is well established that the appropriate a-keto acids giving rise to a particular higher alcohol arise mostly from carbohydrate sources through the synthetic pathways by which yeast synthesizes its amino acid requirements. 

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