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Substitution of Alanine at Position 184 with Glutamic Acid in Escherichia coli PBP5 Ω-Like Loop Introduces a Moderate Cephalosporinase Activity

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Abstract

Escherichia coli PBP5, a DD-carboxypeptidase (DD-CPase), helps in maintaining cell shape and intrinsic β-lactam resistance. Though PBP5 does not have β-lactamase activity under physiological pH, it has a common but shorter Ω-like loop resembling class A β-lactamases. However, such Ω-like loop lacks the key glutamic acid residue that is present in β-lactamases. It is speculated that β-lactamases and DD-CPases might have undergone divergent evolution leading to distinct enzymes with different substrate specificities and functions indicating the versatility of the Ω-loops. Nonetheless, direct experimental evidence favoring the idea is insufficient. Here, aiming to investigate the effect of introducing a glutamic acid residue in the PBP5 Ω-like loop, we substituted A184 to E to create PBP5_A184E. Expression of PBP5_A184E in E. coli ∆PBP5 mutant elevates the β-lactam resistance, especially for cephalosporins. However, like PBP5, PBP5_A184E has the ability to complement the aberrantly shaped E. coli septuple PBP mutant indicating an unaffected in vivo DD-CPase activity. Biochemical and bioinformatics analyses have substantiated the dual enzyme nature of the mutated enzyme possessing both DD-CPase and β-lactamase activities. Therefore, substitution of A184 to E of Ω-like loop alone can introduce the cephalosporinase activity in E. coli PBP5 supporting the phenomenon of a single amino acid polymorphism.

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Acknowledgements

This work is supported by a grant from Department of Biotechnology, Govt. of India (grant # BT/PR8539/BRB/10/1235/2013) to ASG. DK and MD are supported by Indian Institute of Technology Kharagpur. SDP thanks, Science and Engineering Research Board (SERB), Govt. of India for National Post-doctoral Fellowship vide grant # PDF/2015/000011.

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  1. Debasish Kar and Satya Deo Pandey have contributed equally to this work.

Authors and Affiliations

  1. Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India

    Debasish Kar, Satya Deo Pandey, Sathi Mallick, Mouparna Dutta & Anindya S. Ghosh

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  1. Debasish Kar
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  2. Satya Deo Pandey
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Correspondence to Anindya S. Ghosh.

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Kar, D., Pandey, S.D., Mallick, S. et al. Substitution of Alanine at Position 184 with Glutamic Acid in Escherichia coli PBP5 Ω-Like Loop Introduces a Moderate Cephalosporinase Activity. Protein J 37, 122–131 (2018). https://doi.org/10.1007/s10930-018-9765-y

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