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Role of conserved active site tryptophan-101 in functional activity and stability of phosphoserine aminotransferase from an enteric human parasite

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Abstract

Site-directed mutagenesis study was performed to elucidate the role of conserved tryptophan-101 present at the active site of phosphoserine aminotransferase from an enteric human parasite Entamoeba histolytica. Fluorescence resonance energy transfer and molecular dynamic simulation show that the indole ring of Trp101 stacks with the cofactor PLP. Loss of enzymatic activity and PLP polarization values suggest that Trp101 plays a major role in maintaining a defined PLP microenvironment essentially required for optimal enzymatic activity. Studies on W101F, W101H and W101A mutants show that only the indole ring of the conserved Trp101 forms most favorable stacking interaction with the pyridine ring of the cofactor PLP. Protein stability was compromised on substitution of Trp101 with Phe/His/Ala amino acids. A difference in conformational free energy of 1.65 kcal mol−1 was observed between WT-protein and W101A mutant.

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Acknowledgments

In acknowledgement of his formidable biological insights and inspirational mentorship that guided this work VM, AK, VA, KYJZ and TN dedicate this paper to the memory of our co-author, Vinod Bhakuni. We thank Md. Sohail Akhtar for useful discussions and/or comments on the manuscript. VM is thankful to CSIR New Delhi for senior research fellowship. This is communication no. 8136 from CSIR-CDRI.

Conflict of interest

None declared.

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Authors and Affiliations

  1. Division of Molecular and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, 226001, India

    Vibhor Mishra & Vinod Bhakuni

  2. Zhang Initiative Research Unit, Advanced Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan

    Ashutosh Kumar & Kam Y. J. Zhang

  3. Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agam Kuan, Patna, 800007, India

    Vahab Ali

  4. Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-Ku, Tokyo, 162-8640, Japan

    Tomoyoshi Nozaki

Authors
  1. Vibhor Mishra
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  2. Ashutosh Kumar
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  3. Vahab Ali
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  4. Tomoyoshi Nozaki
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  5. Kam Y. J. Zhang
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  6. Vinod Bhakuni
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Corresponding author

Correspondence to Vibhor Mishra.

Additional information

V. Bhakuni: deceased on 15th July 2011.

Electronic supplementary material

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726_2011_1105_MOESM1_ESM.tif

Supplementary Figure 1 A ribbon diagram of EhPSAT dimer showing the position and distance of Trp101 (5.81 Å), Trp47 (26.84 Å) and Trp32 (33.64 Å) with the cofactor PLP for each subunit, respectively. The Trp residues and PLP are shown in blue (with numbers) and red colors, respectively.

726_2011_1105_MOESM2_ESM.tif

Supplementary Figure 2: Distance between PLP and Trp32 (red) and Trp47 (green) throughout molecular dynamics simulation trajectory.

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Mishra, V., Kumar, A., Ali, V. et al. Role of conserved active site tryptophan-101 in functional activity and stability of phosphoserine aminotransferase from an enteric human parasite. Amino Acids 43, 483–491 (2012). https://doi.org/10.1007/s00726-011-1105-x

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  • DOI: https://doi.org/10.1007/s00726-011-1105-x

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