title: Exchanging a single amino acid residue generates or weakens a +2 cellooligosaccharide binding subsite in rice β-glucosidases.
abstract: Os3BGlu6, Os3BGlu7, and Os4BGlu12 are rice glycoside hydrolase family 1 β-glucosidases, the structures of which have been solved by X-ray crystallography. In complex structures, Os3BGlu7 residue Asn245 hydrogen bonds to the second sugar in the +1 subsite for laminaribiose and the third sugar in the +2 subsite for cellotetraose and cellopentaose. The corresponding Os3BGlu6 residue, Met251, appears to block the binding of cellooligosaccharides at the +2 subsite, whereas His252 in this position in Os4BGlu12 could hydrogen bond to oligosaccharides. Mutation of Os3BGlu6 Met251 to Asn resulted in a 15-fold increased k(cat)/K(m) value for hydrolysis of laminaribiose compared to wild type Os3BGlu6 and 9 to 24-fold increases for cellooligosaccharides with degrees of polymerization (DP) of 2-5. On the other hand, mutation of Os3BGlu7 Asn245 to Met decreased the k(cat)/K(m) of hydrolysis by 6.5-fold for laminaribiose and 17 to 30-fold for cellooligosaccharides with DP >2, while mutation of Os4BGlu12 His252 to Met decreased the corresponding k(cat)/K(m) values 2 to 6-fold.
authors.affiliation: School of Biochemistry, Institute of Science, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima 30000, Thailand. sompong_biochem@yahoo.co.th
authors.forename: Sompong
authors.initials: S
authors.lastname: Sansenya
authors.forename: Janjira
authors.initials: J
authors.lastname: Maneesan
authors.forename: James R Ketudat
authors.initials: JR
authors.lastname: Cairns
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism*; D012275:Oryza / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D001617:beta-Glucosidase