Alanine racemases

Fig. 4. Simplified mechanism-based irreversible inactivation of alanine racemase by 3-chlorovinylglycine, P = PO -...

To prevent the formation of byproducts like L-malic add and D-alanine, die cells undergo a pH-treatment to inactive fumarase and alanine racemase. Several reactor conformations have been investigated, but a two reactor system was found to be the most effective. The flow sheet of this two reactor system is given in Figure A8.15. In the first reactor L-aspartic add is formed, which reacts in die second reactor to L-alanine. 

Flynn, G.A., Beight, D.W., Bohme, E.H.W., and Metcalf, B.W., The synthesis of fluorinated aminophosphonic acid inhibitors of alanine racemase, Tetrahedron Lett., 285, 1985. 

Isolated from Streptomyces Amino acid with antimicrobial activity 90 catenulae (inhibitor of alanine racemase)... 

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). 

After formation of the aldimine, numerous factors in the enzyme facilitate deprotonation of the a-carbon (Fig. 3, Step II). The lysine liberated by transimi-nation is utilized as a general base and is properly oriented for effective deprotonation [11]. Furthermore, the inductive effects of the ring system are tuned to increase the stabilization of the quinoid intermediate. For example, the aspartate group that interacts with the pyridyl nitrogen of the co enzyme promotes proto-nation to allow the ring to act as a more effective electron sink. In contrast, in alanine racemase, a less basic arginine residue in place of the aspartic acid is believed to favor racemization over transamination [12]. 

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). 

Cycloserine (Fig- 4) is produced by several species of Streptomyces. One of the basic glycosyl components of the bacterial cell wall, n-acetyl-muramic acid (the product of Mur A and MurB), is modified by the addition of the first three amino acids sequentially by MurC, MurD and MurE enzymes. A dipeptide, D-alanyl-D-alanine is then added to make the pentapeptide. In bacteria, L-alanine is the native form and it is converted to D-alanine form by alanine racemase (Air). Two D-alanines are joined by D-ala-D-ala ligase (DdlA) to synthesize the dipeptide. Cycloserine resembles the substrate for Air and Ddl and inhibits their respective reactions in stage I of the peptidoglycan biosynthesis (Fig. 2). 

PLP-dependent enzymes catalyze the following types of reactions (1) loss of the ce-hydrogen as a proton, resulting in racemization (example alanine racemase), cyclization (example aminocyclopropane carboxylate synthase), or j8-elimation/replacement (example serine dehydratase) (2) loss of the a-carboxylate as carbon dioxide (example glutamate decarboxylase) (3) removal/replacement of a group by aldol cleavage (example threonine aldolase and (4) action via ketimine intermediates (example selenocysteine lyase). 

ALANINE RACEMASE /3-ALANOPINE DEHYDROGENASE d-ALANYL-d-ALANINE PEPTIDASE D-Alanyl-D-alanine synthase,... 

ALANINE RACEMASE S-ALKYLCYSTEINE LYASE ALLIIN LYASE... 

LeMagueres, R Im, H. Dvorak, A. Strych, U. Benedik, M. Krause, K. L. Crystal Structure at 1.45 A Resolution of Alanine Racemase from a Pathogenic Bacterium, Pseudomonas aeruginosa, Contains Both Internal and External Aldimine Forms. Biochemistry 2003, 42, 14752-14761. 

Inhibition of pyridoxal phosphate enzymes by fluoroalanines has been widely studied. Among the numerous examples, alanine racemase, tyrosine phenol... 

Fluoro amino acids have been incorporated into peptides, in order to ease the transport or reduce the systemic toxicity. Thus, trifluoroalanine, a powerful inhibitor of alanine racemase, is an essential enzyme for the biosynthesis of the cell wall of bacteria. It has a low antibiotic activity because of its very poor transport. In order to facilitate this transport, the amino acid has been incorporated into a peptide. This delivery allows a reduction of the doses, and thus the toxicity of the treatment is lowered.3-FIuorophenylaIanine (3-F-Phe) is a substrate of phenylalanine hydroxylase, which transforms it into 3-F-Tyr. 3-F-Tyr has a high toxicity for animals, due to its ultimate metabolization into fluorocitrate, a powerful inhibitor of the Krebs cycle (cf. Chapter 7). 3-F-Phe has a low toxicicity toward fungus cells, but when delivered as a tripeptide 3-F-Phe becomes an efficient inhibitor of the growth of Candida albicans. This tripeptide goes into the cell by means of the active transport system of peptides, where the peptidases set free the 3-F-Phe. ... 

Very detailed studies on the inhibition of alanine racemase by fluoroalanines have been conducted. This enzyme catalyzes the racemization of alanine to provide D-alanine, which is required for synthesis of the bacterial wall. This work has demonstrated that a more complex process than that represented in Figure 7.47 could intervene. For instance, in the case of monofluoroalanine, a second path (Figure 7.48, path b) occurs lysine-38 of the active site can also attack the Schiff base PLP-aminoacrylate that comes from the elimination of the fluorine atom. This enamine inactivation process (path b) has been confirmed by isolation and identification of the alkylation compound, after denaturation of the enzyme (Figure 7.48). ... 

Inhibition processes can highly depend on the number of fluorine atoms. Thus, while mono- and trifluoroalanine are good inhibitors of alanine racemase. 

D-alanine production by alanine racemase, and then preferential excretion of the D-alanine across the cell membrane into the medium. 

Inhibitors of Nucleotide Biosynthesis Suggest mechanisms for the inhibition of (a) alanine racemase by l-fluoroalanine and (b) glutamine amidotransferases by aza-serine. 

Another enzyme-activated inhibitor is the streptomyces antibiotic D-cycloserine (oxamycin), an antitubercular drug that resembles D-alanine in structure. A potent inhibitor of alanine racemase, it also inhibits die non-PLP, ATP-dependent, D-alanyl-D-alanine synthetase which is needed in the biosynthesis of die peptidoglycan of bacterial cell walls. 

Racemization. A proton can be added back to the original alpha position but without stereospecificity. A racemase which does this is important to bacteria. They must synthesize D-alanine and D-glu-tamic acid from the corresponding L-isomers for use in formation of their peptidoglycan envelopes.153-1543 The combined actions of alanine racemase plus D-alanine aminotransferase, which produces D-glutamate as a product, provide bacteria with both d amino acids. 

A fungal alanine racemase is necessary for synthesis of the immunosuppresant cyclosporin (Box 9-F).155 155a High concentrations of free D-alanine are found in certain regions of the brain and also in various glands.156... 

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