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Journal of Bacteriology, July 2003, p. 4211-4218, Vol. 185, No. 14
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.14.4211-4218.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Yoshimitsu Shimomura,1 Yoshimitsu Kakuta,2,3 and Keiichi Fukuyama1,3*

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 {zeta}-crystalline-like soluble quinone oxidoreductase from Thermus thermophilus HB8 (QORTt) and of its complex with NADPH have been determined at 2.3- and 2.8-Å resolutions, respectively. QORTt is composed of two domains, and its overall fold is similar to the folds of Escherichia coli quinone oxidoreductase (QOREc) and horse liver alcohol dehydrogenase. QORTt 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 QORTt was evidenced by circular dichroic measurement. NADPH is located between the two domains in the QORTt-NADPH complex. The disordered segment involved in the coenzyme binding of apo-QORTt 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 QORTt, suggesting that QORTt binds NADPH more readily than NADH. The putative substrate-binding site of QORTt, unlike that of QOREc, is largely blocked by nearby residues, permitting access only to small substrates. This may explain why QORTt has weak p-benzoquinone reduction activity and is inactive with such large substrates of QOREc as 5-hydroxy-1,4-naphthoquinone and phenanthraquinone.

* Corresponding author. Mailing address: Department of Biology, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan. Phone: 81-6-6850-5422. Fax: 81-6-6850-5425. E-mail: fukuyama{at}bio.sci.osaka-u.ac.jp.

Journal of Bacteriology, July 2003, p. 4211-4218, Vol. 185, No. 14
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.14.4211-4218.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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