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J. Biol. Chem., Vol. 277, Issue 37, 34161-34167, September 13, 2002

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Hydroxylation of Indole by Laboratory-evolved 2-Hydroxybiphenyl 3-Monooxygenase^*

Andreas Meyer, Michael Würsten, Andreas Schmid, Hans-Peter E. Kohler^§, and Bernard Witholt^¶

From the  Institute of Biotechnology, Swiss Federal Institute of Technology (ETH) Zurich, ETH Hönggerberg-HPT, CH-8093 Zürich, Switzerland and ^§ Environmental Microbiology and Molecular Ecotoxicology, Swiss Federal Institute of Environmental Sciences and Technology, CH-8600 Dübendorf, Switzerland

Directed enzyme evolution of 2-hydroxybiphenyl 3-monooxygenase (HbpA; EC 1.14.13.44) from Pseudomonas azelaica HBP1 resulted in an enzyme variant (HbpA_ind) that hydroxylates indole and indole derivatives such as hydroxyindoles and 5-bromoindole. The wild-type protein does not catalyze these reactions. HbpA_ind contains amino acid substitutions D222V and V368A. The activity for indole hydroxylation was increased 18-fold in this variant. Concomitantly, the K_d value for indole decreased from 1.5 mM to 78 µM. Investigation of the major reaction products of HbpA_ind with indole revealed hydroxylation at the carbons of the pyrrole ring of the substrate. Subsequent enzyme-independent condensation and oxidation of the reaction products led to the formation of indigo and indirubin. The activity of the HbpA_ind mutant monooxygenase for the natural substrate 2-hydroxybiphenyl was six times lower than that of the wild-type enzyme. In HbpA_ind, there was significantly increased uncoupling of NADH oxidation from 2-hydroxybiphenyl hydroxylation, which could be attributed to the substitution D222V. The position of Asp²²² in HbpA, the chemical properties of this residue, and the effects of its substitution indicate that Asp²²² is involved in substrate activation in HbpA.

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