Recombinant expression, purification, and characterization of ThmD, the oxidoreductase component of tetrahydrofuran monooxygenase |
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Authors: | Michelle Oppenheimer Joshua A Crawford Richard F Helm |
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Institution: | a Department of Biochemistry, Virginia Tech., Blacksburg, VA 24061, USA b Department of Chemistry and Biochemistry, University of Texas at Arlington, TX 76019, USA |
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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. |
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Keywords: | Tetrahydrofuran monooxygenase Oxidoreductase Flavoenzyme Flavin-binding domain Covalent flavin Cytochrome c reductase Iron-sulfur center |
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