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Crystal Structures of the Quinone Oxidoreductase from Thermus thermophilus HB8 and Its Complex with NADPH: Implication for NADPH and Substrate Recognition

Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043,¹ Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581,² RIKEN Harima Institute/SPring-8, Sayo-gun, Hyogo 679-5148, Japan³

Received 28 January 2003/ Accepted 3 April 2003

The crystal structures of the -crystalline-like soluble quinone oxidoreductase from Thermus thermophilus HB8 (QOR_Tt) and of its complex with NADPH have been determined at 2.3- and 2.8-Å resolutions, respectively. QOR_Tt is composed of two domains, and its overall fold is similar to the folds of Escherichia coli quinone oxidoreductase (QOR_Ec) and horse liver alcohol dehydrogenase. QOR_Tt forms a homodimer in the crystal by interaction of the ßF-strands in domain II, forming a large ß-sheet that crosses the dimer interface. High thermostability of QOR_Tt was evidenced by circular dichroic measurement. NADPH is located between the two domains in the QOR_Tt-NADPH complex. The disordered segment involved in the coenzyme binding of apo-QOR_Tt becomes ordered upon NADPH binding. The segment covers an NADPH-binding cleft and may serve as a lid. The 2'-phosphate group of the adenine of NADPH is surrounded by polar and positively charged residues in QOR_Tt, suggesting that QOR_Tt binds NADPH more readily than NADH. The putative substrate-binding site of QOR_Tt, unlike that of QOR_Ec, is largely blocked by nearby residues, permitting access only to small substrates. This may explain why QOR_Tt has weak p-benzoquinone reduction activity and is inactive with such large substrates of QOR_Ec as 5-hydroxy-1,4-naphthoquinone and phenanthraquinone.

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