title: Crystallographic snapshots of an entire reaction cycle for a retaining xylanase from Streptomyces olivaceoviridis E-86.
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.
authors.affiliation: Department of Material and Biological Chemistry, Yamagata University, Japan.
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Suzuki
authors.forename: Zui
authors.initials: Z
authors.lastname: Fujimoto
authors.forename: Shigeyasu
authors.initials: S
authors.lastname: Ito
authors.forename: Shun-Ichi
authors.initials: S
authors.lastname: Kawahara
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kaneko
authors.forename: Kazunari
authors.initials: K
authors.lastname: Taira
authors.forename: Tsunemi
authors.initials: T
authors.lastname: Hasegawa
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kuno
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D020536:Enzyme Activators; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D019810:Sodium Azide; D021241:Spectrometry, Mass, Electrospray Ionization; D013302:Streptomyces / Q000201:enzymology*; D014995:Xylosidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D020536:Enzyme Activators; D050505:Mutant Proteins; D009844:Oligosaccharides; D019810:Sodium Azide; D014995:Xylosidases