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Biochemical Characterization of Inducible ‘Reductase’ Component of Benzoate Dioxygenase and Phthalate Isomer Dioxygenases from Pseudomonas aeruginosa strain PP4

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Abstract

The first step involved in the degradation of phthalate isomers (phthalate, isophthalate and terephthalate) is the double hydroxylation by respective aromatic-ring hydroxylating dioxygenases. These are two component enzymes consisting of ‘oxygenase’ and ‘reductase’ components. Soil isolate Pseudomonas aeruginosa strain PP4 degrades phthalate isomers via protocatechuate and benzoate via catechol ‘ortho’ ring cleavage pathway. Metabolic studies suggest that strain PP4 has carbon source-specific inducible phthalate isomer dioxygenase and benzoate dioxygenase. Thus, it was of interest to study the properties of reductase components of these enzymes. Reductase activity from phthalate isomer-grown cells was 3–5-folds higher than benzoate grown cells. In-gel activity staining profile showed a reductase activity band of R f 0.56 for phthalate isomer-grown cells as compared to R f 0.73 from benzoate-grown cells. Partially purified reductase components from phthalate isomer grown cells showed K m in the range of 30–40 μM and V max = 34–48 μmol min−1 mg−1. However, reductase from benzoate grown cells showed K m = 49 μM and V max = 10 μmol min−1 mg−1. Strikingly similar molecular and kinetic properties of reductase component from phthalate isomer-grown cells suggest that probably the same reductase component is employed in three phthalate isomer dioxygenases. However, reductase component is different, with respect to kinetic properties and zymogram analysis, from benzoate-grown cells when compared to that from phthalate isomer grown cells of PP4.

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Acknowledgment

Research grant from Department of Biotechnology, Govt. of India to PP and Senior research fellowship to RK from IIT-B is gratefully acknowledged.

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  1. Department of Biosciences and Bioengineering, Indian Institute of Technology- Bombay, Powai, Mumbai, 400076, India

    Rohini Karandikar, Abinaya Badri & Prashant S. Phale

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  1. Rohini Karandikar
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  2. Abinaya Badri
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  3. Prashant S. Phale
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Correspondence to Prashant S. Phale.

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Karandikar, R., Badri, A. & Phale, P.S. Biochemical Characterization of Inducible ‘Reductase’ Component of Benzoate Dioxygenase and Phthalate Isomer Dioxygenases from Pseudomonas aeruginosa strain PP4. Appl Biochem Biotechnol 177, 318–333 (2015). https://doi.org/10.1007/s12010-015-1744-6

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