Biochemical characterization and mutational analysis of alanine racemase from Clostridium perfringens

Israr, Muhammad; Lv, Guoping; Xu, Shujing; Li, Yunhe; Ding, Shengting; Zhao, Bin; Ju, Jiansong

doi:10.1016/j.jbiosc.2019.02.003

Cited by 2 publications

(1 citation statement)

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“…Alanine racemase is a key enzyme required for the synthesis of D-alanine, which acts as a pivotal precursor for the biosynthesis of peptidoglycan layers of bacterial cell wall 1 . Hence, its inhibition is found to be fatal to prokaryotes 2,3 . D-cycloserine (DCS) is a broad-spectrum natural drug that interferes with peptidoglycan biosynthesis via the inactivation of the Alanine racemase enzyme.…”

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confidence: 99%

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2023

IJPSR

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Background: In drug design development, Alanine racemase grabbed researchers' attention as it is a potential target for many antimicrobial agents due to its imperative role in bacterial cell wall synthesis. The irresponsible binge eating of antibiotics leads to drug resistance is a key issue of concern for the researchers. The development of multidrug resistance, responsible for causing immense human suffering, demands a renewed attempt to be made to seek antibacterial agents effective against pathogenic bacteria resistant to current antibiotics. This highlights the urgent need for a new and improved antibacterial agent with a new mode of action for use in clinical practice. Method: In the present work, gallic acid derivatives were designed and docked against the active site of the enzyme Alanine racemase (PDB code: 1SFT) using Schrodinger maestro module. Ligands with highest dock score were selected and subjected to ADMET analysis with the help of Qikprop module to study the pharmacokinetic profile of the drug candidate. Result and Conclusion: Based upon computational analysis, compounds (8, 11, 7, 9 and 6) were identified as potential ligands against the binding pockets of Alanine racemase and appeared to be the most promising leads for future drug discovery. ADMET calculation of these compounds falls within the bounds of the satisfactory range without any considerable violation of Lipinski's rule of five. This enables us to infer that these compounds appeared to be the most potential leads that can be investigated as new drug candidates with the improved therapeutic index for further drug design and development.INTRODUCTION: Alanine racemase (EC 5.1.1.1) is an enzyme pervasive in prokaryotic cells and contributes as a therapeutic target for developing antibacterial agents due to its imperative role in bacterial cell wall synthesis. Alanine racemase belongs to the enzymes Isomerases is a pyridoxal-5'-phosphate (PLP) dependent homodimeric enzyme that catalyzes the reversible racemization of L-alanine and Dalanine.

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confidence: 99%

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2023

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Efficient biosynthesis of D/L-alanine in the recombinant Escherichia coli BL21(DE3) by biobrick approach

Naeem,

Hao,

Chu

et al. 2024

Front. Bioeng. Biotechnol.

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Alanine is the most abundant chiral amino acid that exists into the D-alanine or L-alanine forms with diverse applications in the biomedical, pharmaceutical, plastics, and food industries. D/L-alanine production can be carried out through chemical, microbial fermentation, and biocatalytic methods and not much effective due to complicated processes or purification issues and is still challenging to achieve a higher yield. In the present study, biobrick method was utilized for efficient production of D/L-alanine in the recombinant Escherichia coli BL21(DE3) with tandem three-gene co-expression plasmid. Firstly, the co-expression plasmid pET-22bNS-DadX-Ald-Gdh containing three genes, alanine dehydrogenase (ald), alanine racemase (dadX), and glucose dehydrogenase (gdh) from Bacillus pseudofirmus OF4 were successfully constructed and introduced into the E. coli BL21(DE3) strain. Then, under optimized conditions in the whole-cell biocatalytic reaction [20 mM Na2CO3-NaHCO3 (pH 10.1), 200 mM D-glucose, 200 mM sodium pyruvate, and 200 mM ammonium chloride], the concentration of D-alanine and L-alanine reached the maximum value (6.48 g/L and 7.05 g/L) after 3.0 h reaction time at 37°C under 180 rpm rotation. Meanwhile, promoter replacement experiments and Western blot analysis revealed that the expression level of protein OF4Ald had a significant effect on the production of D/L-alanine, indicating that alanine dehydrogenase might be the rate-limiting enzyme for D/L-alanine synthesis. This study provides a simple, feasible, and efficient biosynthesis process of D/L-alanine, which could explore emerging applications for large-scale production of industrial bioproducts.

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Biochemical characterization and mutational analysis of alanine racemase from Clostridium perfringens

Cited by 2 publications

References 32 publications

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Efficient biosynthesis of D/L-alanine in the recombinant Escherichia coli BL21(DE3) by biobrick approach

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