Purification and characterization of arginine deiminase from Pseudomonas putida: Structural insights of the differential affinities of l-arginine analogues

Arginine deiminase (ADI) from Pseudomonas putida was purified using ammonium sulphate precipitation, anion exchange and hydrophobic interaction chromatography. Influence of various chemical compounds (metal ions, reducing agents, sulphydryl agents, and surfactants) on the catalytic activity of ADI w...

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Bibliographic Details
Published in:Journal of bioscience and bioengineering 2019-02, Vol.127 (2), p.129-137
Main Authors: Patil, Mahesh D., Rathod, Vijay P., Bihade, Umesh R., Banerjee, Uttam Chand
Format: Article
Language:English
Subjects:
Arginine deiminase
Homology modeling
Molecular docking
Molecular dynamics
Substrate affinity
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Summary:Arginine deiminase (ADI) from Pseudomonas putida was purified using ammonium sulphate precipitation, anion exchange and hydrophobic interaction chromatography. Influence of various chemical compounds (metal ions, reducing agents, sulphydryl agents, and surfactants) on the catalytic activity of ADI was determined was evaluated on the purified ADI. The enzyme displayed high sensitivity towards thiol binding metal ions, chemicals acting on sulfhydryl group, and most of the surfactants. Substrate specificity studies exhibited that among the eight substrate analogues tested, canavanine had the highest affinity for ADI, followed by d-arginine and guanidine. Canavanine decreased the ADI activity up to 50% at its lowest concentration tested (10 mM), while d-arginine decreased the ADI activity up to ∼4% at its highest tested concentration (200 mM). Differential affinities of the structural analogues of arginine towards ADI were further studied by molecular modeling methods, which included homology modeling, molecular docking and molecular dynamic simulations. The molecular docking studies revealed the critical importance of residues Arg 243, Asp 166, Asp 280, Gly 299 and His 278. RMSDs for protein-ligand complexes were within a range of 1–3 Å, suggesting that the complexes were stable throughout the molecular dynamic simulation. The formation of strong hydrogen bonds by residues Asn 160, Asp166, Arg 185, Arg243, Asp280 and Gly 399 in l-arginine were preserved in the case of d-arginine and canavanine and was responsible for higher affinity towards ADI. Calculations of the substrate binding energies revealed that binding energies ΔGbind and ΔGvdw play a critical role for the differential affinities of various substrate analogues towards P. putida ADI. •Arginine deiminase (ADI) was purified from Pseudomonas putida KT2440.•ADI displayed high sensitivity towards thiol binding metal ions and surfactants.•Canavanine, followed by d-arginine, exhibited the highest affinities for ADI.•The critical importance of residues Arg 243, Asp 166, Asp 280, and His 278 was revealed.•Binding energy components play a critical role for the differential affinities.
ISSN:1389-1723
1347-4421
DOI:10.1016/j.jbiosc.2018.07.021