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Cloning, purification and characterization of two components of phenol hydroxylase from Rhodococcus erythropolis UPV-1

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Abstract

Phenol hydroxylase that catalyzes the conversion of phenol to catechol in Rhodococcus erythropolis UPV-1 was identified as a two-component flavin-dependent monooxygenase. The two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome. The sequenced pheA1 gene was composed of 1,629 bp encoding a protein of 542 amino acids, whereas the pheA2 gene consisted of 570 bp encoding a protein of 189 amino acids. The deduced amino acid sequences of both genes showed high homology with several two-component aromatic hydroxylases. The genes were cloned separately in cells of Escherichia coli M15 as hexahistidine-tagged proteins, and the recombinant proteins His6PheA1 and His6PheA2 were purified and its catalytic activity characterized. His6PheA1 exists as a homotetramer of four identical subunits of 62 kDa that has no phenol hydroxylase activity on its own. His6PheA2 is a homodimeric flavin reductase, consisting of two identical subunits of 22 kDa, that uses NAD(P)H in order to reduce flavin adenine dinucleotide (FAD), according to a random sequential kinetic mechanism. The reductase activity was strongly inhibited by thiol-blocking reagents. The hydroxylation of phenol in vitro requires the presence of both His6PheA1 and His6PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction.

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Acknowledgements

This work was supported by grants from the University of the Basque Country (UPV 42.310-13529/2001 and 42.310-15955/2004). A.J. and L.S. were the recipients of scholarships from the Spanish Ministry of Education and the University of the Basque Country, respectively.

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  1. Enzyme and Cell Technology Group, Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country, P.O. Box 644, 48080, Bilbao, Spain

    Laura Saa, Arrate Jaureguibeitia, Eneko Largo, María J. Llama & Juan L. Serra

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  1. Laura Saa
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  2. Arrate Jaureguibeitia
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  3. Eneko Largo
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  4. María J. Llama
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Correspondence to Juan L. Serra.

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Saa, L., Jaureguibeitia, A., Largo, E. et al. Cloning, purification and characterization of two components of phenol hydroxylase from Rhodococcus erythropolis UPV-1. Appl Microbiol Biotechnol 86, 201–211 (2010). https://doi.org/10.1007/s00253-009-2251-x

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  • DOI: https://doi.org/10.1007/s00253-009-2251-x

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