Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames)

Counago, R.M.; Davlieva, Milya; Strych, Ulrich; Hill, Ryan E.; Krause, Kurt L.

doi:10.1186/1472-6807-9-53

Cited by 32 publications

(44 citation statements)

References 58 publications

(85 reference statements)

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“…As reported earlier, most of the Gram-positive bacteria, including Lactobacillus plantarum (Palumbo et al, 2004), Bacillus anthracis (Couñago et al, 2009), Mycobacterium tuberculosis (Nakatani et al, 2017) and Mycobacterium smegmatis (Chacon et al, 2002), appear to possess only one alanine racemase gene.…”

Section: Discussionsupporting

confidence: 61%

Cloning, Biochemical Characterization and Inhibition of Alanine racemase fromStreptococcus iniae

Muhammad

Gong

et al. 2019

Preprint

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Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species, as well as capable of causing serious disease to humans. Alanine racemase (Alr, EC 5.1.1.1) is a pyridoxal-5′-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-alanine and D-alanine. In this study, we purified alanine racemase from the pathogenic strain of S. iniae, determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The optimal enzyme activity occurred at 35°C and a pH of 9.5. The enzyme belongs to the PLP dependent enzymes family and is highly specific to L-alanine. S.iniae Alr can be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters K m and V max of the enzyme were 33.11 mM, 2426 units/mg for L-alanine and 14.36 mM, 963.6 units/mg for D-alanine.Finally, the 50% inhibitory concentrations (IC 50 ) values and antibiotic activity of two alanine racemase inhibitors, were determined and found to be effective against both gram positive and gram negative bacteria employed in this study. The important role of alanine racemase as a target of developing new antibiotics against S. iniae highlighted the usefulness of the enzyme for new antibiotics discovery.

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Section: Discussionsupporting

confidence: 61%

Cloning, Biochemical Characterization and Inhibition of Alanine racemase fromStreptococcus iniae

Muhammad

Gong

et al. 2019

Preprint

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“…Currently, a variety of amino acid racemases, such as those for alanine, glutamate, serine, aspartate, and arginine, have been discovered in bacteria, archaea, and eukaryotes [23][24][25]. However, only a handful of amino acid racemases that are active with lysine have been isolated.…”

Section: Discussionmentioning

confidence: 99%

Efficient Production of Enantiopure d-Lysine from l-Lysine by a Two-Enzyme Cascade System

Wang

Cao

et al. 2016

Catalysts

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Abstract:The microbial production of D-lysine has been of great interest as a medicinal raw material. Here, a two-step process for D-lysine production from L-lysine by the successive microbial racemization and asymmetric degradation with lysine racemase and decarboxylase was developed. The whole-cell activities of engineered Escherichia coli expressing racemases from the strains Proteus mirabilis (LYR) and Lactobacillus paracasei (AAR) were first investigated comparatively. When the strain BL21-LYR with higher racemization activity was employed, L-lysine was rapidly racemized to give DL-lysine, and the D-lysine yield was approximately 48% after 0.5 h. Next, L-lysine was selectively catabolized to generate cadaverine by lysine decarboxylase. The comparative analysis of the decarboxylation activities of resting whole cells, permeabilized cells, and crude enzyme revealed that the crude enzyme was the best biocatalyst for enantiopure D-lysine production. The reaction temperature, pH, metal ion additive, and pyridoxal 5 -phosphate content of this two-step production process were subsequently optimized. Under optimal conditions, 750.7 mmol/L D-lysine was finally obtained from 1710 mmol/L L-lysine after 1 h of racemization reaction and 0.5 h of decarboxylation reaction. D-lysine yield could reach 48.8% with enantiomeric excess (ee) ≥ 99%.

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“…The unnatural enantiomer of alanine, d ‐Ala, is required in the construction of the peptidoglycan component of the cell wall of bacteria suggesting that an alanine racemase is likely to be essential . The absence of a homolog of alanine racemase in humans has seen this class of enzymes studied intensely as a potential source of a breakthrough in antibacterial research, including in drug resistant strains of C. difficile and against strains of B. anthracis that are potential biological weapons . Indeed, S. coelicolor engineered to lack alanine racemase required the exogenous addition of d ‐Ala.…”

Section: Biological Racemisationmentioning

confidence: 99%

“…[105] The absence of ah omolog of alaniner acemase in humans has seen this class of enzymes studied intensely as a potentials ource of ab reakthrough in antibacterial research,i ncluding in drug resistants trains of C. difficile and against strainso fB .a nthracis that are potentialb iologicalw eapons. [106,107] Indeed, S. coelicolor engineered to lacka laniner acemase required the exogenous addition of d-Ala. Methods to study the racemase activity in S. Lavendulae and E. Coli using circular dichroism have been developed. [105,108] However,a lternative routes for the production of D-Ala take over when alanine racemase is mutated in M. smegmatis [109] and in Chlamydia pneumonia.…”

Section: Enzymes To Catalyse Racemisationmentioning

confidence: 99%

Racemisation in Chemistry and Biology

Ballard

Narduolo

Ahmed

et al. 2020

Chemistry A European J

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The two enantiomers of a compound often have profoundly different biological properties and thus their liability to racemisation in aqueous solutions is an important piece of information. The authors reviewed the available data concerning the process of racemisation in vivo, in the presence of biological molecules (e.g., racemase enzymes, serum albumin, cofactors and derivatives) and under purely chemical but aqueous conditions (acid, base and other aqueous systems). Mechanistic studies are described critically in light of reported kinetic data. The types of experimental measurement that can be used to effectively determine rate constants of racemisation in various conditions are discussed and the data they provide is summarised. The proposed origins of enzymatic racemisation are presented and suggest ways to promote the process that are different from processes taking place in bulk water. Experimental and computational studies that provide understanding and quantitative predictions of racemisation risk are also presented.

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Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames)

Cited by 32 publications

References 58 publications

Cloning, Biochemical Characterization and Inhibition of Alanine racemase fromStreptococcus iniae

Cloning, Biochemical Characterization and Inhibition of Alanine racemase fromStreptococcus iniae

Efficient Production of Enantiopure d-Lysine from l-Lysine by a Two-Enzyme Cascade System

Racemisation in Chemistry and Biology

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