Abstract

The flavin reductase (FRED) and isobutyl amine N-hydroxylase (IBAH) from Streptomyces viridifaciens constitute a two-component, flavin-dependent monooxygenase system that catalyzes the first step in valanimycin biosynthesis. FRED is an oxidoreductase that provides the reduced flavin to IBAH, which then catalyzes the hydroxylation of isobutyl amine (IBA) to isobutyl hydroxylamine (IBHA). In this work, we used several complementary methods to investigate FAD binding, steady-state and rapid reaction kinetics, and enzyme-enzyme interactions in the FRED: IBAH system. The affinity of FRED for FADox is higher than its affinity for FADred, consistent with its function as a flavin reductase. Conversely, IBAH binds FADred more tightly than FADox, consistent with its role as a monooxygenase. FRED exhibits a strong preference (28-fold) for NADPH over NADH as the electron source for FAD reduction. Isothermal titration calorimetry was used to study the association of FRED and IBAH. In the presence of FAD, either oxidized or reduced, FRED and IBAH associate with a dissociation constant of 7-8 μM. No interaction was observed in the absence of FAD. These results are consistent with the formation of a protein-protein complex for direct transfer of reduced flavin from the reductase to the monooxygenase in this two-component system.

Department(s)

Chemistry

Comments

National Science Foundation, Grant 2003658

International Standard Serial Number (ISSN)

1520-4995; 0006-2960

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2024 American Chemical Society, All rights reserved.

Publication Date

12 Jan 2021

PubMed ID

33350810

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