title: Hydroxylation of indole by laboratory-evolved 2-hydroxybiphenyl 3-monooxygenase.
abstract: Directed enzyme evolution of 2-hydroxybiphenyl 3-monooxygenase (HbpA; EC ) 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 microm. 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 Asp(222) in HbpA, the chemical properties of this residue, and the effects of its substitution indicate that Asp(222) is involved in substrate activation in HbpA.
authors.affiliation: Institute of Biotechnology, Swiss Federal Institute of Technology (ETH) Zurich, ETH Hönggerberg-HPT, CH-8093 Zürich, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Meyer
authors.forename: Michael
authors.initials: M
authors.lastname: Würsten
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmid
authors.forename: Hans-Peter E
authors.initials: HP
authors.lastname: Kohler
authors.forename: Bernard
authors.initials: B
authors.lastname: Witholt
mesh_terms: D002384:Catalysis; D006900:Hydroxylation; D007203:Indigo Carmine; D007211:Indoles / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship
chemical_list: D007211:Indoles; D009243:NAD; C030374:indole; D007203:Indigo Carmine; D006899:Mixed Function Oxygenases; C058145:2-hydroxybiphenyl 3-monooxygenase