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Expression, purification, characterization and in silico analysis of newly isolated hydrocarbon degrading bleomycin resistance dioxygenase

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Abstract

In the present investigation, we report cloning, expression, purification and characterization of a novel Bleomycin Resistance Dioxygenase (BRPD). His-tagged fusion protein was purified to homogeneity using Ni-NTA affinity chromatography, yielding 1.2 mg of BRPD with specific activity of 6.25 U mg−1 from 600 ml of E. coli culture. Purified enzyme was a dimer with molecular weight ~ 26 kDa in SDS-PAGE and ~ 73 kDa in native PAGE analysis. The protein catalyzed breakdown of hydrocarbon substrates, including catechol and hydroquinone, in the presence of metal ions, as characterized via spectrophotometric analysis of the enzymatic reactions. Bleomycin binding was proven using the EMSA gel retardation assay, and the putative bleomycin binding site was further determined by in silico analysis. Molecular dynamic simulations revealed that BRPD attains octahedral configuration in the presence of Fe2+ ion, forming six co-ordinate complexes to degrade hydroquinone-like molecules. In contrary, in the presence of Zn2+ ion BRPD adopts tetrahedral configuration, which enables degradation of catechol-like molecules.

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Funding

This research Grant was funded by the Science and Engineering Research Board (SERB) New Delhi, project file number: SB/YS/LS-63/2013 under fast track scheme for the young scientists. Dr PKS would like to thank SERB for financial support.

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Authors and Affiliations

  1. Department of Biotechnology, Sri Guru Granth Sahib World University, Fatehgarh Sahib, Pb, India

    Vinay Sharma & Pushpender Kumar Sharma

  2. Department of Clinical Microbiology, Umeå University, 90185, Umeå, Sweden

    Rajender Kumar

  3. PGI, Chandigarh, India

    Vishal Kumar Sharma & Ashok kumar Yadav

  4. Department of Biological and Environmental Science, Nanoscience Center, University of Jyväskylä, 40014, Jyvaskyla, Finland

    Marja Tiirola

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  1. Vinay Sharma
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Supplementary material 2 (JPEG 61 kb) Supplementary Fig. S2 Secondary structure prediction of BRPD (using I-TASSER)

11033_2019_5159_MOESM2_ESM.jpg

Supplementary material 2 (JPEG 160 kb) Supplementary Fig. S2 Multiple sequence alignment of BRPD and its homologues. The secondary structures of BRPD (QED41507) and two other putative dioxygenases (PDB 3OAJ and 1ZSW) were compared. α helices and β sheets were indicated by red and yellow highlighting respectively. Binding pocket and catalytic residues were indicated in bold. The Fe-co-ordinating residues in the BRPD were indicated with an asterisk

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Sharma, V., Kumar, R., Sharma, V.K. et al. Expression, purification, characterization and in silico analysis of newly isolated hydrocarbon degrading bleomycin resistance dioxygenase. Mol Biol Rep 47, 533–544 (2020). https://doi.org/10.1007/s11033-019-05159-x

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