Hydroxylation of indole by laboratory-evolved 2-hydroxybiphenyl 3-monooxygenase

Abstract

Directed enzyme evolution of 2-hydroxybiphenyl 3-monooxygenase (HbpA; EC 1.14.13.44) from Pseudomonas azelaica HBP1 resulted in an enzyme variant (HbpAind) that hydroxylates indole and indole derivatives such as hydroxyindoles and 5-bromoindole. The wild-type protein does not catalyze these reactions. HbpAind contains amino acid substitutions D222V and V368A. The activity for indole hydroxylation was increased 18-fold in this variant. Concomitantly, the Kd value for indole decreased from 1.5 mM to 78 μM. Investigation of the major reaction products of HbpAind 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 HbpAind mutant monooxygenase for the natural substrate 2-hydroxybiphenyl was six times lower than that of the wild-type enzyme. In HbpAind, 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.