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Protocatechuate 4,5-dioxygenase from Comamonas testosteroni T-2: biochemical and molecular properties of a new subgroup within class III of extradiol dioxygenases

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Abstract

Comamonas testosteroni T-2 degraded at least eight aromatic compounds via protocatechuate (PCA), whose extradiol ring cleavage to 2-hydroxy-4-carboxymuconate semialdehyde (HCMS) was catalysed by PCA 4,5-dioxygenase (PmdAB). This inducible, heteromultimeric enzyme was purified. It contained two subunits, α (PmdA) and β (PmdB), and the molecular masses of the denatured proteins were 18 kDa and 31 kDa, respectively. PCA was converted stoichiometrically to HCMS with an apparent Km of 55 μM and at a maximum velocity of 1.5 μkat. Structure–activity-relationship analysis by testing 16 related compounds as substrate for purified PmdAB revealed an absolute requirement for the vicinal diol and for the carboxylate group of PCA. Besides PCA, only 5′-hydroxy-PCA (gallate) induced oxygen uptake. The N-terminal amino acid sequence of each subunit was identical to the corresponding sequences in C. testosteroni BR6020, which facilitated sequencing of the pmdAB genes in strain T-2. Small differences in the amino acid sequence had significant effects on enzyme stability. Several homologues of pmdAB were found in sequence databases. Residues involved in substrate binding are highly conserved among the homologues. Their sequences grouped within the class III extradiol dioxygenases. Based on our biochemical and genetic analyses, we propose a new branch of the heteromultimeric enzymes within that class.

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Acknowledgements

We are grateful to T. Tralau, who kindly did the Markov analyses, and to D. Schleheck, who did the lutidinic acid experiment. J.M. was funded by the Deutsche Forschungsgemeinschaft (to A.M.C. and J. Ruff) and BASF/BMBF, and M.A.P. by the Alexander von Humboldt Stiftung. Further funds were from the University of Konstanz and the Fonds der Chemischen Industrie.

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  1. Jörg Mampel

    Present address: Institut für Mikrobiologie, Eidgenössische Technische Hochschule Zürich, 8093, Zürich, Switzerland

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  1. Fachbereich Biologie, Universität Konstanz, 78457, Konstanz, Germany

    Jörg Mampel & Alasdair M. Cook

  2. Carleton University, Ottawa, ONT, KIS5B6, Canada

    Miguel A. Providenti

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Mampel, J., Providenti, M.A. & Cook, A.M. Protocatechuate 4,5-dioxygenase from Comamonas testosteroni T-2: biochemical and molecular properties of a new subgroup within class III of extradiol dioxygenases. Arch Microbiol 183, 130–139 (2005). https://doi.org/10.1007/s00203-004-0755-4

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