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Classic Pentachlorophenol Hydroxylating Phenylalanine 4-Monooxygenase from Indigenous Bacillus tropicus Strain AOA-CPS1: Cloning, Overexpression, Purification, Characterization and Structural Homology Modelling

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Abstract

The metabolically promiscuous pentachlorophenol (PCP) hydroxylating Phe4MO (represented as CpsB) was detected, amplified (from the genome of Bacillus tropicus strain AOA-CPS1), cloned, overexpressed, purified and characterized here. The 1.755-kb gene cloned in the pET15b vector expressed a ≅ 64 kDa monomeric protein which was purified to homogeneity by single-step affinity chromatography, with a total yield of 82.1%. The optimum temperature and pH of the enzyme were found to be 30 °C and 7.0, respectively. CpsB showed functional stability between pH 6.0–7.5 and temperature 25–30 °C. The enzyme–substrate reaction kinetic studies showed the allosteric nature of the enzyme and followed pre-steady state using NADH as a co-substrate with apparent vmax, Km, kcat and kcat/Km values of 0.465 μM.s−1, 140 μM, 0.099 s−1 and 7.07 × 10−4 μM−1.s−1, respectively, for the substrate PCP. The in-gel trypsin digestion experiments and bioinformatic tools confirmed that the reported enzyme is a Phe4MO with multiple putative conserved domains and metal ion-binding site. Though Phe4MO has been reported to have a diverse catalytic function, here we report, for the first time, that it functions as a PCP dehalogenase or PCP-4-monooxygenase by hydroxylating PCP. Hence, the use of this enzyme may be further explored in the bioremediation of PCP and other related xenobiotics.

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Acknowledgements

The authors thank Dr Mare Vlok of the CAF, Stellenbosch University, South Africa, for the Liquid-Chromatography Mass-Spectrometry proteomics analysis of the protein.

Funding

National Research Foundation, South Africa (Grant No: 94036 and 92803).

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  1. Discipline of Microbiology, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal (Westville Campus), Private Bag X54001, Durban, 4000, South Africa

    Oladipupo A. Aregbesola, Ajit Kumar, Mduduzi P. Mokoena & Ademola O. Olaniran

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  1. Oladipupo A. Aregbesola
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  2. Ajit Kumar
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  3. Mduduzi P. Mokoena
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  4. Ademola O. Olaniran
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Contributions

O.A. and A.O. conceived and designed the project; O.A. and A.K. designed the experiments; O.A. performed the experiments; M.P. contributed reagents and materials; O.A., A.K., M.P. and A.O. wrote the manuscript; all the authors have read and approved the manuscript.

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Correspondence to Ademola O. Olaniran.

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Aregbesola, O.A., Kumar, A., Mokoena, M.P. et al. Classic Pentachlorophenol Hydroxylating Phenylalanine 4-Monooxygenase from Indigenous Bacillus tropicus Strain AOA-CPS1: Cloning, Overexpression, Purification, Characterization and Structural Homology Modelling. Appl Biochem Biotechnol 194, 635–658 (2022). https://doi.org/10.1007/s12010-021-03645-2

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