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Alanine Racemase


Official Full Name
Alanine Racemase
Background
Mutant Alanine Racemase (Y354N) (mAR-Y354N), a pyridoxal 5-phosphate (PLP) dependent enzyme catalyzes the interconversion of the L-Serine to D-Serine. In WT Alanine Racemase Tyr354 plays a crucial role in defining the strict specificity of AR for alanine, in converting L-Alanine to D-Alanine, which is an important component of the peptidoglycan layer of bacterial cell wall. By mutating the active site Tyr 354 to Asn, the specificity of the enzyme changes and it becomes a racemase with dual specificity for L- Alanine and L-Serine.
Synonyms
Alanine Racemase Y354N; alr; dal; Alanine Racemase

Catalog
Product Name
EC No.
CAS No.
Source
Price
CatalogNATE-1854
EC No.EC 5.1.1.1
CAS No.9024-06-0
SourceE. coli
CatalogNATE-1639
EC No.EC 5.1.1.1
CAS No.
SourceE. coli and fus...
CatalogEXWM-5367
ProductNamealanine racemase
EC No.EC 5.1.1.1
CAS No.9024-06-0
Source
CatalogNATE-0045
EC No.EC 5.1.1.1
CAS No.9024-06-0
SourceBacillus stearo...
Related Reading

Alanine racemase (Alr), also known as L-alanine racemase, is a ubiquitous prokaryotic enzyme. This enzyme belongs to the family of isomerases that act on amino acids and their derivatives, and participates in the metabolism of alanine and aspartate. Alanine racemase utilizes pyridoxal 5’-phosphate (PLP) to perform the racemization of L-alanine and provide D-alanine for cell wall synthesis. The enzyme can be found throughout bacteria, which makes alanine racemase an attractive target for antibacterial drug development, but in some bacteria, there are alternative pathways for D-alanine synthesis, so alanine racemase will not serve as antibacterial targets for these bacteria. In eukaryotes such as aquatic animals, fungi and plants, few similar enzymes are found. The genome sequence of methanogenic archaeon, Methanococcus maripaludis shows that the alanine dehydrogenase gene is adjacent to the alanine racemase and alanine permease. In addition to its function in the utilization of D-alanine, Alr can also protect the system from D-alanine inhibition because the growth of Alr mutants on the mixture of D and L-alanine was compromised. Recent studies have shown that in Chlamydia pneumoniae lacking genes encoding alanine racemase and DadX homologs, serine hydroxymethyl transferase GlyA can be used as a source of D-alanine.

Structure of alanine racemase

Many crystal structures of Alr and inhibitor complexes have been resolved. One of the best-studied structures is Alr (PDB ID: 1SFT) from Geobacillus stearothermophiluswhich reveals a 388-residue homodimeric enzyme formed by a head-to-tail association of two monomers. Each monomer is composed of two folded domains: (i) the N-terminal domain formed by 1-240aa, and (ii) the C-terminal domain formed by 241-388aa.

The N-terminal domain is composed of eight-stranded α/β-barrel, and the C-terminal domain is composed of only the β-strands. Molecular dynamics studies have shown that there are water molecules in the two active sites of Alr homodimers. Since this water molecule plays an important role, it should be fully considered when designing inhibitors. The active site in each monomer of Alr contains pyridoxal 5'-phosphate (PLP) binding residues covalently linked to Lys39, as well as amino acids in the immediate environment of the pyridoxal cofactor. Lys39 is located at the C-terminus of the first β chain of the α/β-barrel, and its side chain points to the center of the barrel. The angle at which the catalytic domains of each monomer are inclined to each other is 125°. Therefore, the Lys39 residue also extends in the cleft formed by the two domains of the monomer, while the PLP moiety positions itself in the approximate center of the barrel, closer to the second domain of the other monomer. 

Pictorial view of the Bacillus stearothermophilus Alr homodimer (PBD ID: 1SFT) Figure 1. Pictorial view of the Bacillus stearothermophilus Alr homodimer (PBD ID: 1SFT) (Azam, M.A.; Jayaram, U. 2016)

Inhibitors of alanine racemase

The search for Alr enzyme inhibitors began in 1966 when Lynch and Neuhaus explained the mechanism of action of O-carbamoyl-D-serine in Streptococcus faecalis. In this study, O-carbamoyl-D-serine was found to be an effective Alr inhibitor and was supported by enzyme kinetic studies. The focus of the research is to establish the primary action site of o-carbamoyl-d-serine on Alr, and compared with other action sites such as D-ala-D-ala synthetase, D-ala-D-ala adding enzyme, and phospho-NAc-muramyl-pentapeptide translocase.

It was also observed that 200 μg/mL D-isomer had no effect on Streptococcus lactis, Lactobacillus arabinosus, and E. coli, these results are consistent with those reported by Neuhaus and Lambert in 1972. They also proved that L-cycloserine is an effective competitive inhibitor, and previously, L-cycloserine was considered to be ineffective for the inhibition of Staphylococcus aureus Alr. Atherton et al. synthesized the L-alanine analog Alafosfalin, which was found to be a selective inhibitor of peptidoglycan biosynthesis in both Gram-positive and Gram-negative bacteria. It increases the accumulation of diphosphate-N-acetyl-muramyl-tripeptide in gram-positive organisms and reduces the intracellular levels of D-alanine. In bacteria, alafosfalin is transported by stereospecific peptide permeases, and is finally hydrolyzed into 1-aminoethyl phosphonic acid by intracellular aminopeptidases. The target site of 1-aminoethyl phosphonic acid is the Alr enzyme, which is reversibly and competitively inhibited in Gram-negative E. coli and irreversibly inhibits Gram-positive such as Staphylococcus aureus and Staphylococcus faecalis in a time-dependent manner.

Structure of Alr inhibitor O-carbamyl-D-serine and alafosfalin Figure 2. Structure of Alr inhibitor O-carbamyl-D-serine and alafosfalin (Azam, M.A.; Jayaram, U. 2016)

References

  1. Azam, M.A.; Jayaram, U. Inhibitors of alanine racemase enzyme: a review. Journal of Enzyme Inhibition and Medicinal Chemistry. 2016.
  2. Atherton, F.R.; et al. Phosphonopeptides as antibacterial agents: mechanism of action. Antimicrob Agents Chemother. 1979, 15: 696-705.

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