Transaminases reaction scheme

In addition to the identification of the appropriate biosynthetic enzyme, the feasibility of all biotransformation processes depends heavily on other criteria, such as the availability of inexpensive starting materials, the reaction yield, and the complexity of product recovery, hi the case of transaminase processes, the reversible nature of the reaction (Scheme 3.1) and the presence of a keto acid by-product is a concern that limits the overall yield and purity of product and has led to efforts to increase the conversion beyond the typical 50% yield of product.99 100 Additionally, there are cost considerations in the large-scale preparation of keto acid substrates such as 2-ketobutyrate, which are not commodity chemicals. 

Although the utility of transaminases has been widely examined, one such limitation is the fact that the equilibrium constant for the reaction is near unity. Therefore, a shift in this equilibrium is necessary for the reaction to be synthetically useful. A number of approaches to shift the equilibrium can be found in the literature [37,85.90]. Another method to shift the equilibrium is a modification of that previously described. Aspartate is used as the amino donor, which is converted into oxaloacetate (25) upon reaction (Scheme 17). Since 25 is unstable, it decomposes to pyruvate (26) and thus favors product formation. However, since pyruvate is itself an a-keto acid, it must be removed, or else it will be converted into alanine, which could cause downstream processing problems. This is accomplished by inclusion of the alsS gene encoding for the enzyme acetolac... 

This removal of the reaction by-product has been achieved through the use of aspartic acid as the amino donor (Scheme 16). The amine group transfer results in the fonnation of oxaloacetate (7), an unstable compound that decarboxylates under the reaction conditions to afford pyruvate (8). As 8 is still an a-keto acid, and is a substrate for a transaminase reaction that results in the production of alanine, another enzyme is used to dimerize the pyruvate. The product of this reaction is acetolactate (9), which, in turn, spontaneously undergoes decarboxylation to result in the overall formation of acetoin (10) as the final by-product. Acetoin is simple to remove and does not participate in any further reactions. Thus, the equilibrium is driven to provide the desired unnatural amino acid that makes the isolation straightforward. 

Transaminases will be specific for a-amino acids of one configuration and during the reaction (Schemes 1 and 6), conformation 3a will be transformed to the imine 4. This can be protonated either from the Si face (as in 4d) or the Re face to give 5 and thence pyridoxamine 6. 

It has since been shown that reversible, non-enzymic transamina- tion occurs between pyridoxal and alanine and pyridoxamine and pyruvate in neutral, aqueous solutions, at 25° and in the absence of metal ions. (No evidence has yet been obtained for the presence rfj metal ions in purified mammalian transaminases.) Under thesi f conditions, no decarboxylation or racemisation of the amino add could be detected. From the results of kinetic and spectrophotometricl studies in this simple model system for transamination it has beenf shown that imines are formed rapidly and reversibly between the twtf pairs of reactants and that tautomerisation of the two SchifF s bascf intermediates is rate limiting in the overall transamination reactioni The reaction scheme is given in Figure 7. 

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). 

The aminotransferase class of enzymes (E.C. 2.6.1.x), also known as transaminases, are ubiquitous, PLP-requiring enzymes that have been used extensively to prepare natural L-amino acids and other chiral compounds.30 123 124 The L-aminotransferases catalyze the general reaction shown in Scheme 19.19 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 (see also Chapter 3). Those enzymes most commonly used as industrial biocatalysts have been cloned, overexpressed, and generally used as whole-cell or immobilized preparations. These include branched chain aminotransferase (BCAT) (E.C. 2.6.1.42), aspartate aminotransferase (AAT) (E.C. 2.6.1.1), and tyrosine aminotransferase (TAT) (E.C. 2.6.1.5). 

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. 

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. 

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). - " " ... 

This process, for example, can be nsed for the prodnction of both l- and D-2-aminobntyrate (30 and 31, respectively) (Scheme 9.29 and Scheme 9.30). Other than for the transaminase enzyme, the reactions cascade is the same for by-prodnct removal for both enantiomers. 

A modified cyclodextrin (25), which contains a pyridoxamine moiety, catalyzes the transamination as shown in Scheme 8 [39]. The reaction between (25) and indolepyruvic acid (26) is 200 times faster than the reaction between pyridoxamine and (26), since the first reaction is an intracomplex one and the second is an intermolecular one. Transamination is completed by the reaction of (27), formed by the reaction between (25) and (26), with another amino acid, regenerating (25). Thus (25) which consists of a cyclodextrin and a pyridoxamine is a model of the enzyme transaminase. 

Transaminases (also termed amino transferases [EC 2.6.l.X]) catalyze the redox-neutral amino-transfer reaction between an amine donor and a carbonyl group serving as acceptor (Scheme 2.225) [94, 1707-1712]. These enzymes require an activated benzaldehyde (pyridoxal-5 -phosphate, PLP, vitamin Bg) as cofactor, which functions as a molecular shuttle for the transfer of the NHa-moiety. In a first step, PLP forms a ketimine Schiff base with the amine-donor. Tautomerization of the C=N bond yields an aldimine, which is hydrolyzed to yield the cofactor in its aminated form (pyridoxamine, PMP). The latter reacts through the same order of events with the carbonyl group of the substrate to form the amine product and... 

A great deal of early research in the field established the foundations that now govern the concept of enzyme mimicry using MIPs, including the importance of structure-function relationships in determining binding and catalytic activity. Nicholls etal. prepared and evaluated an MIP transaminase mimic for the reaction of phenylpyruvic acid and pyridoxamine (Scheme 22). ... 

In 2012, Kroutil and co-workers reported the first amination reaction of primary alcohols with ammonium chloride by an artificial multi-enzyme-catalyzed cascade method (Scheme 29) [173]. The authors assumed that the reaction might proceed by two steps. Initially, the alcohol was oxidized by an alcohol dehydrogenase (ADH), consuming NAD" " and leading to the formation of the aldehyde and NADH. Then, the aldehyde intermediate was aminated with an amine donor L-alanine by a w-transaminase (w-TA). Finally, by combining ADH-hT (ADH from Bacillus stearothermophilus) with CV-w-TA (w-TA from Chromobacterium violaceuni), the amination of various primary alcohols successfully afforded the corresponding primary amines in 2-99 % yields. 

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