Transaminases amino acid

There is a further possibility for conversion of methionine to MT. Transaminase enzymes can convert methionine to 4-methylthio-2-oxobutanoate (also, a-keto-y-methylthiobutyrate). One such enzyme is aromatic-amino-acid transaminase, EC 2.6.1.57, for which L-methionine is, albeit, less efficiently, a substrate (Equation 5) ... 

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 ... 

Turning to l-AAO, Pantaleone s industrial research group have reported" on the properties and use of an l-AAO from Proteus myxofaciens, overexpressed in Escherichia coli This l-AAO, unusually, appears not to produce H2O2 in the catalytic reaction, thus making the addition of catalase unnecessary. The enzyme has a broad specificity, with a preference for nonpolar amino acids. This l-AAO was used in conjunction with a D-amino acid transaminase (d-AAT) and an alanine racemase (AR) to allow an efficient conversion of L-amino acid in to D-amino acid (Scheme 4). 

EC2.6.1.57 aromatic L-amino acid transaminase (aminotransferase (transaminase))... 

This enzyme [EC 2.6.1.21], also known as D-aspartate aminotransferase, D-amino acid aminotransferase, and D-amino acid transaminase, catalyzes the reversible pyridoxal-phosphate-dependent reaction of D-alanine with a-ketoglutarate to yield pyruvate and D-glutamate. The enzyme will also utilize as substrates the D-stereoisomers of leucine, aspartate, glutamate, aminobutyrate, norva-hne, and asparagine. See o-Amino Acid Aminotransferase... 

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]. 

Another useful transaminase, D-amino acid transaminase (DAT) (E.C. 2.6.1.21), has been the subject of much study.53 133 134 This enzyme catalyzes the reaction using a D-amino acid donor, either alanine, aspartate, or glutamate (Scheme 19.20), to produce another D-amino acid. 

So far no Cys residue has been demonstrated to be involved in AspAT functions. Is this a common phenomenon among transaminases Merola et al,67 attempted to address the role of Cys residues in D-amino acid transaminase by site-directed mutagenesis and found that none of the Cys residues was essential for the catalysis. At the moment, it appears that no Cys residue is catalytically involved in pyridoxal-dependent transaminases. 

For two transaminases the remaining unknown stereochemical parameter was determined by demonstrating an internal transfer of tritium (dialkyl amino acid transaminase) [28] or deuterium (pyridoxamine-pyruvate transaminase) [27] from the a-position of the substrate L-alanine to C-4 of the cofactor. Internal hydrogen transfer from the a-position of the substrate amino acid to C-4 of PLP has also been demonstrated for two of the abortive transamination reactions, those catalyzed by tryptophan synthase fi2 protein [32] and by aspartate-/8-decarboxylase [31]. In addition, the same phenomenon must occur in alanine transaminase, as deduced from the observation that the enzyme catalyzes exchange of the /8-hydrogens of... 

Scheme VI. Stereochemical possibilities in the reduction of an L-amino acid-transaminase equilibrium complex with tritiated NaBH4.

When the amino donor has the L-configuration and an L-amino acid transaminase is used, the product amino acid will have an L-configuration and when the amino donor has the D-configuration and a D-amino transaminase is used, the product amino acid will have a D-configuration. The equilibrium constant for the reaction is typically close to unity. 

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. 

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). 

Another example is provided by r>-tert-leucine (4) (Scheme 10), where an asymmetric approach cannot be used. This unnatural amino acid is one of the few that cannot be made by our current o-amino acid transaminase. While this enzymatic method is being researched, small amounts of material have been prepared by a resolution method [24]. 

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. 

We have previously described the isolation and cloning of genes encoding microbial L-amino acid transaminases [40], and the use of site-directed mutagenesis to enhance enzyme function [41]. The reversibility of the reaction has frequently been considered a drawback of transaminase commercial application because it results in reduced yields and complicates product isolation. Through molecular cloning we have constructed bacterial strains that effectively eliminate this concern by removal of the keto acid by-product, and thereby significantly increase the reaction yield. 

D-Amino acid transaminases have been less well characterized but proceed by a similar catalytic mechanism and show similar potential as effective biocatalysts. Further strain development has incorporated amino acid racemases, enabling complete utilization of racemic amino donors. Thus, L-aspartic acid can be used as the n-amino acid donor through use of aspartate racemase within the system. 

Transamination reactions combine reversible amina-tion and deamination, and they mediate redistribution of amino groups among amino acids. Transaminases (aminotransferases) are widely distributed in human tissues and are particularly active in heart muscle, liver, skeletal muscle, and kidney. The general reaction of transamination is... 

The Stickland reaction (47) has received much attention as a possible route to chiral acetate due to the availability of chiral glycine (48). In the Stickland reaction two moles of glycine and one mole of d-alanine are converted quantitatively into three moles of acetate, three moles of ammonia, and one mole of C02 by the organism Clostridium sticklandii. The presence of amino acid transaminase in the intact organisms leads to extensive hydrogen exchange although in the purified enzyme the replacement of NH2 by H occurs stereospecifically with inversion (49, 50). Unfortunately, the rates of conversion with the purified enzyme are too low to be synthetically useful. 

Transaminases are generally not considered to be enzymes catalyzing redox reactions, which is obvious considering the meaning of the E. C. code for transferases (E. C. 2.6.1.x = transferring amino groups). Nevertheless, the exchange of an amino functionality between an amino acid and an a-keto acid implies the oxidation of the amino acid. Transaminases are described elsewhere in this book (Chapter 12). 

D-Amino acid transaminases (DATs) are also available and catalyze the analogous reaction to Scheme 9.27 but need a n-amino acid donor (alanine, aspartate, or glutamate) to produce another D-amino acid (Scheme 9.28). - " " ... 

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