Enzyme amino acid racemases

Several hundred tons of L-methionine per year are produced by enzymatic conversion in an enzyme membrane reactor. An alternative approach is dynamic resolution, where the unconverted enantiomer is racemized in situ. Starting from racemic /V-acetyl-amino acid, the enantioselective L-acylase is used in combination with an TV-acyl-amino acid racemase to enable nearly total conversion of the substrate. 

In Section 5.03.6.2, a stereoselective synthesis of L-homophenylalanine from the racemic AAacetylated amino acid is described. The authors, however, found that substrate solubility limited the utility of this procedure. Having found an L-N-carbamoylase in Bacillus kaustophilus, they introduced the gene for this enzyme together with that for the N-acyl amino acid racemase from D. radiodurans into E. coli for coexpression. These cells, permeabilized with 0.5% toluene, were able to deliver L-homophenylalanine in 99% yield and were able to be used for multiple reaction cycles. 

The reversal of this process could potentially occur with reprotonation from either face of the C=N double bond, and a mixture of aldimines would result, leading to generation of a racemic amino acid. This accounts for the mode of action of PLP-dependent amino acid racemase enzymes. Of course, the enzyme controls removal and supply of protons this is not a random event. One important example of this reaction is alanine racemase, employed by bacteria to convert L-alanine into o-alanine for cell-wall synthesis (see Box 13.12). 

Among the numerous enzymes that utilize pyridoxal phosphate (PLP) as cofactor, the amino acid racemases, amino acid decarboxylases (e.g., aromatic amino acids, ornithine, glutamic acid), aminotransferases (y-aminobutyrate transaminase), and a-oxamine synthases, have been the main targets in the search for fluorinated mechanism-based inhibitors. Pharmaceutical companies have played a very active role in this promising research (control of the metabolism of amino acids and neuroamines is very important at the physiological level). 

T Although D-amino acids do not generally occur in proteins, they do serve some special functions in the structure of bacterial cell walls and peptide antibiotics. Bacterial peptidoglycans (see Fig. 20-23) contain both D-alanine and D-glutamate. D-Amino acids arise directly from the l isomers by the action of amino acid racemases, which have pyridoxal phosphate as cofactor (see Fig. 18-6). Amino acid racemization is uniquely important to bacterial metabolism, and enzymes such as... 

The alanine racemization catalyzed by alanine racemase is considered to be initiated by the transaldimination (Fig. 8.5).26) In this step, PLP bound to the active-site lysine residue forms the external Schiff base with a substrate alanine (Fig. 8.5, 1). The following a-proton abstraction produces the resonance-stabilized carbanion intermediates (Fig. 8.5, 2). If the reprotonation occurs on the opposite face of the substrate-PLP complex on which the proton-abstraction proceeds, the antipodal aldimine is formed (Fig. 8.5,3). The subsequent hydrolysis of the aldimine complex gives the isomerized alanine and PLP-form racemase. The random return of hydrogen to the carbanion intermediate is the distinguishing feature that differentiates racemization from reactions catalyzed by other pyridoxal enzymes such as transaminases. Transaminases catalyze the transfer of amino group between amino acid and keto acid, and the reaction is initiated by the transaldimination, followed by the a-proton abstraction from the substrate-PLP aldimine to form a resonance-stabilized carbanion. This step is common to racemases and transaminases. However, in the transamination the abstracted proton is then tranferred to C4 carbon of PLP in a highly stereospecific manner The re-protonation occurs on the same face of the PLP-substrate aldimine on which the a-proton is abstracted. With only a few exceptions,27,28) each step of pyridoxal enzymes-catalyzed reaction proceeds on only one side of the planar PLP-substrate complex. However, in the amino acid racemase... 

Amino acid racemases are important for bacteria because they need D-alanine in the biosynthesis of cell walls. These enzymes require pyridoxal as the active cofactor. A racemization reaction starts with the aldimine complex between pyridoxal and an a-amino acid (Scheme 2.4). Deprotonation occurs at the a-carbon of amino acid, due to the electron-sink effect of pyridoxal. Reprotonation of the quinonoid intermediate at the opposite side provides the desired product (pathway a in Scheme 2.4). However, reprotonation may also take place at the C4 of pyridoxal (pathway b in Scheme 2.4). This kills the catalyst because one of its product, pyridoxamine, can no longer racemize an amino acid. 

For several reasons a-amino acids are ideal substrates for deracemization methods. They racemize easily by base catalysis under a number of conditions and they are racemized in Nature by the intervention of specific amino acid racemases. They are also recognized as substrates by oxidative enzymes to give the corresponding oxo-acids, in turn substrates for amino transferases and amino acid dehydrogenases. Several industrial preparations of L- and D-amino acids are based on processes of deracemization [26] or of separate two-steps resolution-racemization [27]. 

Several inventive procedures have been used for overcoming these difficulties, offering interesting multistep enzyme-catalyzed reactions for deracemization of amino acids. The requirement of an amino donor of D-configuration can be solved by its in situ generation from the L-form by using an amino acid racemase. 

Enzymatic Kinetic Resolution of N-Acyl Amino Acids Coupled with Racemization by N-Acyl Amino Acid Racemase Acylases are enzymes hydrolysing the N-acetyl derivatives of amino acids. They require the free carboxylate for activity and have long been used for the kinetic resolution of amino acids. The unreacted enantiomer is usually racemized in a separate step by treatment with acetic anhydride. While acylases from hog kidney have an L-specificity, bacterial acylases with L- and D-specificity of various origins have been isolated and used for the kinetic resolution of N-acetyl amino acids. An industrial process for the production of L-Met and other proteinogenic and non-proteinogenic L-amino acids such as L-Val, L-Phe, L-Norval, or L-aminobutyric acid has been established. Currently, several hundred tons per year of L-methionine are produced by this enzymatic conversion using an enzyme membrane reactor [46]. 

A variety of amino acid racemases have been identified in bacteria, archaea, and eukaryotes. They are dassified into two groups pyridoxal 5 -phosphate (PLP) -dependent and -independent enzymes. Therefore, racemization can be achieved via two mechanisms through a chiraUy unstable Schiff base intermediate with an aromatic aldehyde serving as co-factor PLP (Scheme 13.22a) and by a two-base mechanism without co-factor (Scheme 13.22b). 

The glycolytic pathway includes three such reactions glucose 6-phosphate isomer-ase (1,2-proton transfer), triose phosphate isomerase (1,2-proton transfer), and eno-lase (yS-elimination/dehydration). The tricarboxylic acid cycle includes four citrate synthase (Claisen condensation), aconitase (j5-elimination/dehydration followed by yS-addition/hydration), succinate dehydrogenase (hydride transfer initiated by a-proton abstraction), and fumarase (j5-elimination/dehydration). Many more reactions are found in diverse catabolic and anabolic pathways. Some enzyme-catalyzed proton abstraction reactions are facilitated by organic cofactors, e.g., pyridoxal phosphate-dependent enzymes such as amino acid racemases and transaminases and flavin cofactor-dependent enzymes such as acyl-C-A dehydrogenases others. 

An amino acid racemase which shows very broad substrate specificity was discovered in Pseudomonas striata (= Ps. putida), purified, and characterized1 91. The enzyme catalyzes racemization of various amino acids except aromatic and acidic... 

Rudnick and Abeles purified proline racemase to 95% homogeneity from Clostridium sticklandii, and characterized it 92. The enzyme is composed of two identical subunits with a molecular weight of about 38000, and is independent of any cofactors or metals. Most amino acid racemases require pyridoxal 5 -phosphate, which labilizes the bond between the a-hydrogen and the chiral center by aldimine formation with the a-amino group of the substrate. However, PLP is not involved in the reaction of proline racemase acting on an a-imino acid. The enzyme also acts on 2-hydroxy-L-proline and 2-allo-hydroxy-D-proline although slowly they are epimer-ized at a rate of 2 and 5% of the rate of L-proline racemization, respectively. L-Proline and D-proline showed Km values of 2.9 and 2.5 mti, respectively1119. ... 

The N-acetyl-D,L-amino acid precursors are conveniently accessible through either acetylation of D,L-amino acids with acetyl chloride or acetic anhydride in a Schotten-Baumann reaction or via amidocarbonylation I801. For the acylase reaction, Co2+ as metal effector is added to yield an increased operational stability of the enzyme. The unconverted acetyl-D-methionine is racemized by acetic anhydride in alkali, and the racemic acetyl-D,L-methionine is reused. The racemization can also be carried out in a molten bath or by an acetyl amino acid racemase. Product recovery of L-methionine is achieved by crystallization, because L-methionine is much less soluble than the acetyl substrate. The production is carried out in a continuously operated stirred tank reactor. A polyamide ultrafiltration membrane with a cutoff of 10 kDa retains the enzyme, thus decoupling the residence times of catalyst and reactants. L-methionine is produced with an ee > 99.5 % and a yield of 80% with a capacity of > 3001 a-1. At Degussa, several proteinogenic and non-proteinogenic amino acids are produced in the same way e.g. L-alanine, L-phenylalanine, a-amino butyric acid, L-valine, l-norvaline and L-homophenylalanine. 

AlaR, the bacterial enzyme that catalyzes the racemization of l- and D-alanine, is the most investigated PLP-dependent amino acid racemase. It is also the only PLP-dependent racemase whose structure has been solved. 

A variety of other enzymes involved in amino acid catabolism have been detected in both protozoa and helminths. These include deaminases such as histidase, decarboxylases, some of which are involved in biosynthesis of amines and related compounds, and hydroxylases of proline, tryptophan and tyrosine. These additional enzymes have mostly been reported in helminths (1). L-Amino acid oxidases and D-amino acid oxidases are also present and the availability of the latter would allow D-amino acids to be metabolized in the absence of amino acid racemases. 

Amino acid racemases catalyse the formation of a racemic mixture from either l- or D-amino acids. The enzymes of this class have only been detected in bacteria where they are involved in the formation of D-amino acids required for cell-wall biosynthesis. A crucial step in the racemase catalysed reactions must involve the cleavage of C -H bond to give a quinonoid intermediate of type 2 (Fig. 10) which is then protonated in a non-stereospecific fashion. Detailed mechanistic studies on these... 

The recycling of the undesired enantiomer from the enzymatic resolution is of crucial importance particularly on an industrial scale [107]. The classical chemical method consists of the thermal racemization of an amino acid ester at about 150-170°C. Milder conditions can be employed for the racemization of the corresponding amides via intermediate formation of Schiff bases with aromatic aldehydes such as benzaldehyde or salicylaldehyde (Scheme 2.14). More recently, intense research has been devoted to the use of isomerase enzymes (such as amino acid racemases [108]) aiming at the development of dynamic resolution processes. 

Enzymatic racemisation is an attractive option in DKR because the reactions catalysed by enzymes are performed under mild conditions. The Degussa group have recently described their successful commercialization of two DKR-based processes that employ racemases, namely (i) the DKR of 5-substituted hydantoins using whole cells coexpressing a L-carbamoylase, a hydantoin racemase and a hydantoinase and (ii) the DKR of N-acetyl amino acids using an acylase in combination with an N-acetyl amino acid racemase from Amycolatopsis orientalis. 

Cells of strain 10716 cultured in environments containing less than 6 X 10 M Mn+2 are unable to synthesize detectable quantities of bacitracin (Weinberg and Tonnis, 1966). When 2 X 10" M Mn+ is supplied to such cultures during the maximum stationary phase, the peptide appears in detectable concentrations in culture supernatants within four hr. However, if a quantity of chloramphenicol sufficient to suppress protein synthesis but not to kill vegetative cells is added either simultaneously or within two hr following the addition of Mn+, bacitracin is not formed. Apparently, in cells deprived of the metalhc ion, synthesis of either amino acid racemases or peptide assembly enzymes is suppressed (Weinberg and Tonnis, 1966). 

L-a-Amino acids have been prepared by the resolution of racemic a-amino acid amide by the L-specific aminopeptidase from Pseudomonas putida ATCC 12633 [7]. Enzyme from R putida ATCC 12633 cannot be used to resolve a-alkyl-substituted amino acid amides 103. Aminoamidase from Mycobacterium neoaurum ATCC 25795 has been used in the preparation of L-a-alkyl amino acid 104 (Fig. 34) and D-amide of a-alkyl-substituted amino acids by enzjmaatic resolution process using racemic a-alkyl amino acid amide as a substrate [169,179]. Amidase from Ochrobactrum anthropi catalyzed the resolution of a,a-disubsituted amino acids, iV-hydroxy amino acids, and a-hydroxy acid amides. The resolution process could lead to the production of chiral amino acids or amides in 50% yield. Recently, amino acid racemases have been used to get 100% yield of chiral amino acids [179]. 

Asano et al. have developed an approach for the synthesis of D-amino acids through DKR using a two-enzyme system [55]. They had previously reported the discovery of new D-stereospecific hydrolases that can be applied to KR of racemic amino acid amides to yield D-amino acids. Combination of a D-stereospedfic hydrolase with an amino acid amide racemase allows performing DKR of i-amino acid amides yielding enantiomerically pure D-amino acids in excellent yields (Figure 4.29). 

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