title: Heterologous expression, purification, crystallization and preliminary X-ray analysis of Trichoderma reesei xylanase II and four variants.
abstract: Xylanase II from Trichoderma reesei catalyzes the hydrolysis of glycosidic bonds in xylan. Crystallographic studies of this commercially important enzyme have been initiated to investigate its reaction mechanism, substrate binding and dependence on basic pH conditions. The wild-type protein was heterologously expressed in an Escherichia coli host using the defined medium and four active-site amino acids were replaced to abolish its activity (E177Q and E86Q) or to change its pH optimum (N44D and N44H). Cation-exchange and size-exclusion chromatography were used to obtain >90% protein purity. The ligand-free proteins and variant complexes containing substrate (xylohexaose) or product (xylotriose) were crystallized in several different space groups and diffracted to high resolutions (from 1.07 to 1.55 Å).
authors.affiliation: Biology and Soft Matter Division, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN 37831, USA.
authors.forename: Qun
authors.initials: Q
authors.lastname: Wan
authors.forename: Andrey
authors.initials: A
authors.lastname: Kovalevsky
authors.forename: Qiu
authors.initials: Q
authors.lastname: Zhang
authors.forename: Scott
authors.initials: S
authors.lastname: Hamilton-Brehm
authors.forename: Rosalynd
authors.initials: R
authors.lastname: Upton
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Weiss
authors.forename: Marat
authors.initials: M
authors.lastname: Mustyakimov
authors.forename: David
authors.initials: D
authors.lastname: Graham
authors.forename: Leighton
authors.initials: L
authors.lastname: Coates
authors.forename: Paul
authors.initials: P
authors.lastname: Langan
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity; D014242:Trichoderma / Q000737:chemistry* / Q000201:enzymology; D014312:Trisaccharides / Q000737:chemistry* / Q000378:metabolism; D014990:Xylans / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D014312:Trisaccharides; D014990:Xylans; C515044:xylotriose; D043364:Endo-1,4-beta Xylanases
keywords: Trichoderma reesei; xylanase II