Pyridoxal alanine racemase

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

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... 

Like modular PKSs, peptide synthetases also epimerize some substrates and/or intermediates. For example, the starter substrate amino acid of cyclosporin A is D-Ala. Racemization of alanine is not catalyzed by an integrated subunit of cyclosporin A synthetase, but by alanine racemase. This is a separate, pyridoxal phosphate-dependent enzyme [ 193]. In contrast, Grsl and Tycl covalently activate L-Phe as a thioester and subsequently epimerize the amino acid [194]. D-Phe is the only epimer accepted as a substrate for dipeptide formation by Grs2 and Tyc2 [195, 196]. No racemization activity is detected in a pantetheine-deficient mutant of Grsl [197]. Deletion mutagenesis pointed to the requirement of the COOH-terminal part of the module for epimerizing L-Phe to D-Phe [180]. In contrast, the biosynthesis of actinomycin D, a bicyclic chromo-pentapeptide lactone (Fig. 10), involves formation of the dipeptide 6-MHA (methylanthranilic acid)-L-Thr-L-Val prior to epimerization of the L-Val exten-... 

Perhaps the best characterized organic cofactor-dependent racemase is alanine racemase, which employs pyridoxal 5 -phosphate (PLP) (Table 7.1). o-alanine is necessary for the synthesis of the peptidoglycan layer of bacterial cell walls in Gram negative and positive bacteria [1]. Alanine racemase is thus a ubiquitous enzyme in bacteria and an excellent drug target [2]. Both its crystal structure and mechanism have been well investigated. PLP reacts with amino acids to produce... 

Alanine racemase is a bacterial enzyme that catalyzes racemization of l- and d-alanine, and requires pyridoxal 5 -phosphate (PLP) as a cofactor. The enzyme plays an important role in the bacterial growth by providing D-alanine, a central molecule in the peptidoglycan assembly and cross-linking, and has been purified from various sources15 161. The enzyme has been used for the production of stereospecifically deuterated NADH and various D-amino acids by combination of L-alanine dehydrogenase (E. C. 1.4.1.1), D-amino acid aminotransferase (E. C. 2.6.1.21), and formate dehydrogenase (E.C. 1.2.1.2)I17, 18. ... 

Chloroalanine has been found to be an irreversible inhibitor of the pyridoxal phosphate-linked yS-aspartate decarboxylase/ aspartate aminotransferase/ and alanine racemase. The mechanism of inhibition is shown above by Eq. (7) (the sulfate reacts in the same manner) amino-ethane sulfonate irreversibly inhibits pyridoxal phosphate-linked GABA transaminase and L-serine-O-sulfate irreversibly inhibits aspartate aminotransferase. ... 

The existence of enzymes in microorganisms which catalyze the interconversion of D- and L-amino acids is of considerable interest, since the intramolecular transfer of an amino group is apparently involved. The term racemase has been proposed for such enzymes. Two racemases have been reported. Alanine racemase has been shown to be present in a large number of microorganisms and has been partially purified from extracts of S. faecalis. Glutamic acid racemase has been demonstrated in acetone powders of Lactobacillus arabinosus. Both enzymes catalyze the interconversion of the n- and l- forms of their respective substrates. Alanine racemase requires pyridoxal phosphate as coenzyme. Pyri-doxamine phosphate under the conditions employed was not active. Glutamic acid racemase was found not to be affected by the addition of pyridoxal phosphate. However, further studies with purified preparations are necessary before pyridoxal phosphate can be excluded as cofactor for the glutamic acid racemase. Examination of animal tissues under conditions favorable for the demonstration of bacterial alanine racemase failed to reveal any activity. 

The Cofactor Pyridoxal 5 -phosphate from Organic Models to Alanine Racemase and Aspartate Aminotransferase... 

Chan-Huot, M., Dos, A., Zander, R., Sharif, S., Tolstoy, P.M., Compton, S., Fogle, E., Toney, M.D., Shenderovich, I.G., Denisov, G.S., and Limbach, H.H. (2013) NMR studies of protonation and hydrogen bond states of internal aldimines of pyridoxal 5 -phosphate acid-base in alanine racemase, aspartate aminotransferase and poly-L-lysine. / Am. Chem. Soc., 135, 18160-18175,... 

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... 

D-Alanine is found in bacterial cell wall peptidogly-can. l-Alanine is converted to D-alanine by a racemase that contains pyridoxal phosphate as a cofactor. The racemiza-tion is followed by the formation of a D-alanyl-D-alanine dipeptide, which is accompanied by the conversion of ATP to ADP. The dipeptide is subsequently incorporated into the glycopeptide (see fig. 16.16). 

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. 

Racemases are enzymes capable of interconverting D- to L-amino acids. Pyridoxal phosphate has been claimed to play a role as a cofactor in bacterial racemases for alanine, glutamic acid, and methionine, but not in others. It has also been claimed that in mammals the administration of pyridoxine facilitates the use of D-amino acids. 

Scheme 12.2. The reversible transamination of aspartate (Asp, D) and alanine (Ala, A) as catalyzed by aminotransferase using pyridoxal as a cofactor. In principle (and apparently in practice), the same process can be used to convert any a-ketocarboxyhc acid to the corresponding amino acid. The a-proton is picked up and deposited on the same side (unless a racemase is involved) of the two amino acids. EC numbers and some graphic materials provided in this scheme have been taken from appropriate hnks in a URL starting with http // www.chem.qmul.ac.uk/iubmb/enzyme/.

In certain bacteria there is a specific nutritional requirement for D-amino acids which are found as components of cell structures or antimetabolites. Bacteria normally meet this need by the conversion of L-amino acids to D-amino acids and in the case of alanine, methionine and tryptophan the evidence suggests that these reactions are directly catalysed by amino acid racemases which have a cofactor requirement for pyridoxal phosphate . An oxidation-reduction cofactor may also be a general feature of racemases of this class. However, the mode of epimerisation of L-phenylalanine to D-phenylalanine necessary for the synthesis of some peptide antibiotics, proceeds in an entirely different way, which as yet has only been partially resolved. 

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