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Journal of Biochemistry Advance Access originally published online on March 11, 2009
Journal of Biochemistry 2009 146(1):61-70; doi:10.1093/jb/mvp047
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Crystallographic Snapshots of an Entire Reaction Cycle for a Retaining Xylanase from Streptomyces olivaceoviridis E-86

Ryuichiro Suzuki1,2, Zui Fujimoto2, Shigeyasu Ito1, Shun-Ichi Kawahara3,*, Satoshi Kaneko4, Kazunari Taira3,5,6,7, Tsunemi Hasegawa1 and Atsushi Kuno8,{dagger}

1Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560; 2Protein Research Unit, National Institute of Agrobiological Sciences, Tsukuba 305-8602; 3Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8562; 4 Food Biotechnology Division, National Food Research Institute, Tsukuba 305-8642; 5Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Tokyo 113-8656; 6Tokyo University and Graduate School of Social Welfare, Isesaki, Gunma 372-0831; 7Nagase & Co., Ltd, Nihonbashi, Tokyo 103-8355; and 8Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8568, Japan

{dagger}To whom correspondence should be addressed. Tel: +81-298613187, Fax: +81-298613125, E-mail: atsu-kuno{at}aist.go.jp

Received December 7, 2008; Accepted February 28, 2009


  Abstract

Retaining glycosyl hydrolases, which catalyse both glycosylation and deglycosylation in a concerted manner, are the most abundant hydrolases. To date, their visualization has tended to be focused on glycosylation because glycosylation reactions can be visualized by inactivating deglycosylation step and/or using substrate analogues to isolate covalent intermediates. Furthermore, during structural analyses of glycosyl hydrolases with hydrolytic reaction products by the conventional soaking method, mutarotation of an anomeric carbon in the reaction products promptly and certainly occurs. This undesirable structural alteration hinders visualization of the second step in the reaction. Here, we investigated X-ray crystallographic visualization as a possible method for visualizing the conformational itinerary of a retaining xylanase from Streptomyces olivaceoviridis E-86. To clearly define the stereochemistry at the anomeric carbon during the deglycosylation step, extraneous nucleophiles, such as azide, were adopted to substitute for the missing base catalyst in an appropriate mutant. The X-ray crystallographic visualization provided snapshots of the components of the entire reaction, including the E•S complex, the covalent intermediate, breakdown of the intermediate and the enzyme–product (E•P)complex.

Key Words: chemical rescue, crystallographic visualization, molecular evolution, retaining GH10 β-xylanase, switching enzyme with azide

Abbreviations: Cel5A, GH 5 cellulase from Bacillus agaradhaerens; BSUA, {alpha}-amylase from Bacillus subtilis; CGTase, cyclodextrin glycosyltransferase from Bacillus circulans strain 251; Cel12A, GH 12 cellulase from Humicola grisea; Cel7B, GH 7 cellulase from Fusarium oxysporum; DNP2Fcell, 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-D-cellobioside; Thio-DP5, Methyl 4',4'',4'''-S-trithio-{alpha}-cellotetraoside; SoXyn10A, GH10 xylanase from Streptomyces olivaceoviridis E-86; pNP-X2, p-Nitrophenyl-β-D-xylobioside; MU-X2, 4-methylumbelliferyl-β-D-xylobioside; X5, xylo-pentasaccharide; LBHB, low-barrier hydrogen bond; CEX, GH10 xylanase from Cellulomonas fimi; ESI-MS, electrospray ionization mass spectrometry

*Deceased December 1, 2004.


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