Branched-chain amino acid transaminase

Transaminase enzymes (also called aminotransferases) specifically use 2-oxoglutarate as the amino group acceptor to generate glutamate but some have a wide specificity with respect to the amino donor. For example, the three branched-chain amino acids leucine, isoleucine and valine, all serve as substrates for the same enzyme, branched-chain amino acid transaminase, BCAAT ... 

Removal of the amino groups by branched-chain amino acid transaminase forms the corresponding a-keto acids. 

As discussed earlier, the avermectin polyketide backbone is derived from seven acetate and five propionate extender units added to an a branched-chain fatty acid starter, which is either (S( I )-a-mcthylbutyric acid or isobutyric acid. The C25 position of naturally occurring avermectins has two possible substituents a. sec-butyl residue derived from the incorporation of S(+)-a-methy lbutyry 1-CoA ( a avermectins), or an isopropyl residue derived from the incorporation of isobutyiyl-CoA ( b avermectins). These a branched-chain fatty acids, which act as starter units in the biosynthesis of the polyketide ring, are derived from the a branched-chain amino acids isoleucine and valine through a branched-chain amino acid transaminase reaction followed by a branched-chain a-keto acid dehydrogenase (BCDH) reaction (Fig. 5) [23]. 

Lflly, M., Bauer, F.F., Styger, G., Lambrechts, M.G., Pretorius, l.S. (2006b).The effect of increased branched-chain amino acid transaminase activity in yeast on the production of higher alcohols and on the flavour profiles of wine and distillates. FEMS Yeast Res., 6, 726-743. 

Dehydration by CymH putatively yielded 2-hydroxy-5-methylhexa-2,4,-dienoic acid 182. Methylation by the SAM-dependent MT CymG followed by transamination utilizing a branched-chain amino acid transaminase would provide ADH (Figure 3.58) [187]. 

Finally, transamination with the branched-chain amino acid transaminase (EC 2.6.1.42), the coenzyme for which is pyridoxal and the source of the amino group from which pyridoxal transfers the nitrogen is glutamate (Glu, E), occurs. 2-Oxoglutarate forms in the process. 

Scheme 12.16. A representation of the final stages in the biosynthesis of isoleucine (He, I). In the late stages shown here, the migration of the ethyl group is accompanied by the reduction of the carbonyl generated in that migration (the enzyme is a reductoisomerase (EC 1.1.1.86). Dehydration to an enol (a dihydroxy acid dehydratase, EC4.2.1.9), tautomeriza-tion to the corresponding ketone, and a final transamination ( branched-chain amino acid transaminase with pyridoxal as a cofactor) from glutamate (Glu,E) produces isoleuciue (He, I). EC numbers and some graphic materials provided in this scheme have been taken from appropriate links in a URL starting with http //www.chem.qmul.ac.uk/iubmb/enzyme/.

Branched-chain amino acid glutamate transaminase... 

Glucocorticoids also increase the activity of transaminases (aminotransferases), especially in the skeletal muscle. Aminotransferases serve to transfer the amino groups from amino acids to be metabolized to a-keto acids, especially pyruvate. In the latter case, the alanine thus formed is transported from the muscle into the bloodstream and extracted from there by the liver. In the liver, alanine is converted to glucose, and glucose may then return to the muscle as it does in the Cori cycle (Figure 18.4). This is the alanine cycle, and more about this is discussed in Chapter 20. Branched-chain amino acids are the principal donors of nitrogen to pyruvate in the muscle and are thus important actors in the alanine cycle. 

Branched Chain Amino Acids valine (val), leucine (leu), and isoleucine (ilu). The metabolism of each of these three amino acids begins with the same theme transaminase DH Complex foeta-oxidation. Due to the irreversible nature of the DH Complex all three are essential. 

FIGURE 12.1 The Ehrlich pathway exemplified for the conversion of the branched-chain amino acids L-valine, L-isoleucine, and L-leucine to the corresponding alcohols isobntanol, 2-methyl-1-bntanol, and 3-methyl-1-butanol. Adh, alcohol dehydrogenase KE)C, 2-ketoacid decarboxylase TA, transaminase. 

Rudman and Meister IJ ) first showed the presence of a transaminase in cell-free extracts of E. colt that catalyze transamination reactions between glutamate and isoleucine, valine, leucine, norleucine, and norvaline. These monocarboxylic amino acids transaminated with each other as well as with glutamine. Preparations of an E. cdi mutant which did not respond to a-keto- 8-methylvalerate was unable to transaminate isoleucine or valine. The transaminase responsible for activity with the branched-chain amino acids was separated from other transaminases and considerably purified by standard methods of protein purification. It was shown to... 

The three branched chain amino acids are normally metabolized as shown in Figure 6.2. Each amino acid is converted to the corresponding Of-keto acid by a transaminase specific for that amino acid. A solitary case of valinaemia is known, caused by lack of valine transaminase [76] the patient is mentally retarded. The three a-keto acids are decarboxylated by two (or possibly three) enzyme systems, one specific for a-keto-isovaleric acid, the other acting on a-keto-isocaproic and Q -keto-j3-methylvaleric acids [77, 78]. The reaction is complex, proceeding in three distinct steps [78] and requiring coenzyme A, thiamine pyrophosphate, lipoic acid and NAD. The end products are the co-enzyme A thio-esters of the branched chain fatty acids. 

Transaminases possess many features appropriate for effident biocatalysts, such as high turnover numbers and no requirement for external recycling of the co-factor. Because of the wide substrate tolerance of many amino transferases such as tyrosine amino transferase and branched-chain amino transferases from E. coU, these enzymes have been largely employed in the enantiospecific preparation of non-proteinogenic amino acids. These include straight-chain alkyl, diadd, branched-chain, aromatic, and bifunctional amino adds [65]. 

The aminotransferase, or transaminase class of enzymes, are ubiquitous, PLP-requiring enzymes that have been used extensively to prepare natural L-amino acids [84,85]. They catalyze the general reaction shown in Scheme 15, where an amino group from one L-amino acid is transferred to an a-keto acid to produce a new L-amino acid and the respective a-keto acid. Those enzymes most commonly used have been cloned, overexpressed, and generally used as whole cell or immobilized preparations. These include the following branched chain aminotransferase (SCAT) (EC 2.6.1.42), aspartate aminotransferase (AAT) (EC 2.6.1.1), and tyrosine aminotransferase (TAT) (EC 2.6.1.5). A transaminase patented by Celgene Corporation (Warren. NJ), called an co-aminotransferase, does not require an a-amino acid as amino donor and hence is used to produce chiral amines [86,87]. Another useful transaminase, n-amino acid transaminase (DAT) (EC 2.6.1.21), has been the subject of much study [37,88,89]. This enzyme catalyzes the reaction using a n-amino acid donor, either alanine or aspartate (Scheme 16). 

L-Amino acid transaminases are ubiquitous in nature and are involved, be it directly or indirectly, in the biosynthesis of most natural amino acids. All three common types of the enzyme, aspartate, aromatic, and branched chain transaminases require pyridoxal 5 -phosphate as cofactor, covalently bound to the enzyme through the formation of a Schiff base with the e-amino group of a lysine side chain. The reaction mechanism is well understood, with the enzyme shuttling between pyridoxal and pyridoxamine forms [39]. With broad substrate specificity and no requirement for external cofactor regeneration, transaminases have appropriate characteristics to function as commercial biocatalysts. The overall transformation is comprised of the transfer of an amino group from a donor, usually aspartic or glutamic acids, to an a-keto acid (Scheme 15). In most cases, the equilibrium constant is approximately 1. 

Importantly, its precise mechanism of its anticonvulsant action is still not fully understood. However, it has been duly advocated that its administration specifically inhibits GABA-transaminase, and thereby enhancing the concentration of cerebral GABA. It has also been observed that a few other straight-chain saturated fatty acids i.e., lower fatty acids, such as propanoic acid, butyric acid, and pentanoic acid which are devoid of anticonvulsant characteristic features are relatively more potent and efficacious inhibitors of GABA-transaminase than is valproic acid. Furthermore, it has been adequately substantiated that there exists a rather stronger correlation between the anticonvulsant potency of valproate and other branched-chain fatty acids besides, their capability to minimise the prevailing concentration of cerebral aspartic acid (an amino acid). 

A possible explanation for the superiority of the amino donor, L-aspartic add, has come from studies carried out on mutants of E. coli, in which only one of the three transaminases that are found in E. coli are present. It is believed that a branched chain transaminase, an aromatic amino add transaminase and an aspartate phenylalanine aspartase can be present in E. coli. The reaction of each of these mutants with different amino donors gave results which indicated that branched chain transminase and aromatic amino add transminase containing mutants were not able to proceed to high levels of conversion of phenylpyruvic add to L-phenylalanine. However, aspartate phenylalanine transaminase containing mutants were able to yield 98% conversion on 100 mmol l 1 phenylpyruvic acid. The explanation for this is probably that both branched chain transaminase and aromatic amino acid transminase are feedback inhibited by L-phenylalanine, whereas aspartate phenylalanine transaminase is not inhibited by L-phenylalanine. In addition, since oxaloacetate, which is produced when aspartic add is used as the amino donor, is readily converted to pyruvic add, no feedback inhibition involving oxaloacetate occurs. The reason for low conversion yield of some E. coli strains might be that these E. cdi strains are defident in the aspartate phenylalanine transaminase. 

There are several examples of d to l inversion of amino acids in the literature. D-Phenylalanine may have therapeutic properties in endogenous depression and is converted to L-phenylalanine in humans [145]. o-Leucine is inverted to the L-enantiomer in rats. When o-enantiomer is administered, about 30% of the enantiomer is converted to the L-enantiomer with a measurable inversion from l to o-enantiomer. As indicated in Fig. 13, D-leucine is inverted to the L-enantiomer by two steps. It is first oxidized to a-ketoisocarproate (KIC) by o-amino acid oxidase. This a-keto acid is then asymmetrically reaminated by transaminase to form L-leucine. In addition, KIC may be decarboxylated by branched-chain a-keto acid dehydrogenase, resulting in an irreversible loss of leucine (Fig. 13) [146]. D-Valine undergoes a similar two-step inversion process, and this can be antagonized by other amino acids such as o-leucine. The primary factor appears to be interference with the deamination process [147]. 

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