Amino acid racemases substrate specificity

The one-base mechanism is characterized by the retention of the substrate-derived proton in the product (internal retum).30) With this criterion, reactions catalyzed by a-amino-c-caprolactam racemase,323 amino acid racemase of broad specificity from Pseudomonas striata333 have been considered to proceed through the one-base mechanism. However, such internal returns were not observed in the reactions of alanine racemases from K coli B,33) B. stearothermophilus,263 and S. typhirmaium (DadB and /1/r).263 The internal return should not be observed in the two-base mechanism, because the base catalyzing the protonation to the intermediate probably obtains the proton from the solvent. But the failure of the observation of the internal return can be also explained by the single-base mechanism in which exchange of the proton abstracted from the substrate a-carbon with the solvent is much faster than its transfer to the a-carbanion. Therefore, lack of the internal return does not directly indicate the two-base mechanism of the alanine racemase reaction. 

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]. 

Amino Acid Racemase with Low Substrate Specificity (E.C. 5.1.1.10)... 

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... 

Amino acid racemase with low substrate specificity catalyzes racemization of leucine and various other amino acids, which are also a-deuterated in 2H20 during their racemization[63). Therefore, [4S-2H]-NADH was produced in the same manner as described above with the racemase and L-leucine dehydrogenase (E. C. 1.4.1.9), which is pro-S specific[35). 

Makiguchi and coworkers established a method to synthesize L-tryptophan from D,L-serine and indole by means of tryptophan synthase (E.C. 4.2.1.20) from E. coli and the amino acid racemase with low substrate specificity of Ps. striata (= Ps. putida) I65L Both D,L-serine and indole are cheaply available by chemical synthesis. Tryptophan synthase catalyzes the (3-replacement reaction of L-serine with indole to produce L-tryptophan, and the amino acid racemase with low substrate specificity converts unreacted D-serine into L-serine. Because the racemase does not act on tryptophan, almost all d,L-serine is converted into optically pure L-tryptophan. Makiguchi et al.[65] succeeded in producing L-tryptophan in a 200 L reactor using intact cells of E. coli and Ps. putida 65. Under the optimal conditions established, 110 g L 1 of L-tryptophan was formed in molar yields of 91 and 100% for added d,l-serine and indole, respectively, after 24 h of incubation with intermittent indole feeding. Continuous production of L-tryptophan was also achieved using immobilized cells of E. coli and Ps. putida. The maximum concentration of L-tryptophan formed was 5.2 g L 1 (99% molar yield for indole). 

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]. 

Pyruvate is transaminated with hypotaurine by ca-ainino acid aminotransferase to form alanine, and acetaldehyde and sulinate, which are formed irreversibly from sulfinoace-toaldehyde and primarily produced [4R- H]NADH in a high yield. In contrast, [4S- H]NADH is produced with LeuDH (pro-S stereospecific) and amino acid racemase with low substrate specificity in a sunilar manner. [4S- H]NADPH can also be synthesized by means of NADP-dependent GluDH (pro-S stereosj ific) and glutamate racemase in a similar method [93]. 

Racemic a-amino amides and a-hydroxy amides have been hydrolyzed enantio-selectively by amidases. Both L-selective and o-selective amidases are known. For example, a purified L-selective amidase from Ochrobactrum anthropi combines a very broad substrate specificity with a high enantioselectivity on a-hydrogen and a,a-disubstituted a-amino acid amides, a-hydroxyacid amides, and a-N-hydroxya-mino acid amides [102]. A racemase (a-amino-e-caprolactam racemase, EC 5.1.1.15) converts the o-aminopeptidase-catalyzed hydrolysis of a-amino acid amides into a DKR (Figure 6.38) [103]. 

In many cases, the racemization of a substrate required for DKR is difficult As an example, the production of optically pure cc-amino acids, which are used as intermediates for pharmaceuticals, cosmetics, and as chiral synfhons in organic chemistry [31], may be discussed. One of the important methods of the synthesis of amino acids is the hydrolysis of the appropriate hydantoins. Racemic 5-substituted hydantoins 15 are easily available from aldehydes using a commonly known synthetic procedure (Scheme 5.10) [32]. In the next step, they are enantioselectively hydrolyzed by d- or L-specific hydantoinase and the resulting N-carbamoyl amino acids 16 are hydrolyzed to optically pure a-amino acid 17 by other enzymes, namely, L- or D-specific carbamoylase. This process was introduced in the 1970s for the production of L-amino acids 17 [33]. For many substrates, the racemization process is too slow and in order to increase its rate enzymes called racemases are used. In processes the three enzymes, racemase, hydantoinase, and carbamoylase, can be used simultaneously this enables the production of a-amino acids without isolation of intermediates and increases the yield and productivity. Unfortunately, the commercial application of this process is limited because it is based on L-selective hydantoin-hydrolyzing enzymes [34, 35]. For production of D-amino acid the enzymes of opposite stereoselectivity are required. A recent study indicates that the inversion of enantioselectivity of hydantoinase, the key enzyme in the... 

A substantial number of PLP enzymes catalyze the racemization or epimerization of primary a-amino acids [54,55]. Of particular physiological importance are microbial alanine racemases because of their involvement in bacterial cell wall formation, which makes them a potential target for chemotherapy. An interesting substrate specificity is exhibited by diaminopimelate racemase [56] which acts only on meso-and LL-diaminopimelate, but not on the DD-isomer, i.e., the enzyme requires the L configuration at one end of the molecule in order to epimerize the chiral center at the other end. Racemization is also occasionally observed as an alternate catalytic... 

The enzymes of the nucleic acid metabolism are used for several industrial processes. Related to the nucleobase metabolism is the breakdown of hydantoins. The application of these enzymes on a large scale has recently been reviewed [85]. The first step in the breakdown of hydantoins is the hydrolysis of the imide bond. Most of the hydantoinases that catalyse this step are D-selective and they accept many non-natural substrates [78, 86]. The removal of the carbamoyl group can also be catalysed by an enzyme a carbamoylase. The D-selective carbamoylases show wide substrate specificity [85] and their stereoselectivity helps improving the overall enantioselectivity of the process [34, 78, 85]. Genetic modifications have made them industrially applicable [87]. Fortunately hydantoins racemise readily at pH >8 and additionally several racemases are known that can catalyze this process [85, 88]. This means that the hydrolysis of hydantoins is always a dynamic kinetic resolution with yields of up to 100% (Scheme 6.25). Since most hydantoinases are D-selective the industrial application has so far concentrated on D-amino acids. Since 1995 Kaneka Corporation has produced 2000 tons/year of D-p-hydroxyphenylglycine with a D-hydantoinase, a d-carbamoylase [87] and a base-catalysed racemisation [85, 89]. 

The product of a NHase/amidase cascade reaction is an acid, which is the same as the single enzymatic reaction performed by a nitrilase. However, the NHases usually have different substrate specificities than nitrilases, making them more suitable for the production of certain compounds. Although most organisms have both NHase and amidase activity (see earlier text), it is sometimes preferable, in a synthetic application, to combine enzymes from different organisms. The reasons for this are the enantioselectivity of the amidase or specific activity or substrate specificity of either of the enzymes. In this way, products with different enantiomeric purity can be obtained. Recently, a coupling of a NHase with two different amidases with opposite enantiopreference together with an -amino-a-caprolactam racemase that allows the formation of small aliphatic almost enantiopure (R)- or (S)-amino acids via dynamic kinetic resolution processes has been described [52]. 

Specific activity for L-2-amino butyric acid amide was 9.5U/mg, which was 2.7% that for l-ACL (350U/mg with a substrate concentration of 100 mM). Activities toward alanine amide, threonine amide, norvaline amide, and norleucine amide were all <2.1% that for ACL. The enzyme did not act on a-amino acid, peptides consisting of alanine, or alanine methyl esters. The values for 2-aminobutyramide and alanine amide were both calculated to be 1.0, which identified them as typical racemase-catalyzed reactions [23] ... 

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