Recombinant expression, purification, and characterization of ThmD, the oxidoreductase component of tetrahydrofuran monooxygenase.
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 463nm and a small peak at 396 nm. Computational modeling and mutagenesis analyses suggest a novel three-dimensional fold or covalent flavin attachment in ThmD.
Department of Biochemistry, Virginia Tech., Blacksburg, VA 24061, USA.
Michelle
M
Oppenheimer
Brad S
BS
Pierce
Joshua A
JA
Crawford
Keith
K
Ray
Richard F
RF
Helm
Pablo
P
Sobrado
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; C433514:tetrahydrofuran monooxygenase