Recombinant Expression, Purification, and Characterization of ThmD, the Oxidoreductase Component of Tetrahydrofuran Monooxygenase

Abstract

Tetrahydrofuran monooxygenase (Thm) catalyzes the NADH-and oxygen-dependent hydroxylation of tetrahydrofuran to 2-hydroxytetrahydrofuran. Thm is composed of a hydroxylase enzyme, a regulatory subunit, and an oxidoreductase named ThmD. ThmD was expressed in Escherichia coli as a fusion to maltose-binding protein (MBP) and isolated to homogeneity after removal of the MBP. Purified ThmD contains covalently bound FAD, [2Fe-2S] center, and was shown to use ferricyanide, cytochrome c, 2,6-dichloroindophenol, and to a lesser extent, oxygen as surrogate electron acceptors. ThmD displays 160-fold preference for NADH over NADPH and functions as a monomer. The flavin-binding domain of ThmD (ThmD-FD) was purified and characterized. ThmD-FD displayed similar activity as the full-length ThmD and showed a unique flavin spectrum with a major peak at 463. nm and a small peak at 396. nm. Computational modeling and mutagenesis analyses suggest a novel three-dimensional fold or covalent flavin attachment in ThmD. © 2010.

Department(s)

Chemistry

Keywords and Phrases

Covalent flavin; Cytochrome c reductase; Flavin-binding domain; Flavoenzyme; Iron-sulfur center; Oxidoreductase; Tetrahydrofuran monooxygenase

International Standard Serial Number (ISSN)

1096-0384; 0003-9861

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2024 Elsevier, All rights reserved.

Publication Date

01 Apr 2010

PubMed ID

20159007

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