title: Active site cleft mutants of Os9BGlu31 transglucosidase modify acceptor substrate specificity and allow production of multiple kaempferol glycosides.
abstract: BACKGROUND
  Rice Os9BGlu31 is a transglucosidase that can transfer glucose to phenolic acids, flavonoids, and phytohormones. Os9BGlu31 displays a broad specificity with phenolic 1-O-β-D-glucose esters acting as better glucose donors than glucosides, whereas the free phenolic acids of these esters are also excellent acceptor substrates.
  
  
  METHODS
  Based on homology modeling of this enzyme, we made single point mutations of residues surrounding the acceptor binding region of the Os9BGlu31 active site. Products of the wild type and mutant enzymes in transglycosylation of phenolic acceptors from 4-nitrophenyl β-D-glucopyranoside donor were identified and measured by UPLC and negative ion electrospray ionization tandem mass spectrometry (LCMSMS).
  
  
  RESULTS
  The most active variant produced was W243N, while I172T and L183Q mutations decreased the activity, and other mutations at W243 (A, D, M, N, F and Y) had variable effects, depending on the acceptor substrate. The Os9BGlu31 W243N mutant activity was higher than that of wild type on phenolic acids and kaempferol, a flavonol containing 4 hydroxyl groups, and the wild type Os9BGlu31 produced only a single product from each of these acceptors in significant amounts, while W243 variants produced multiple glucoconjugates. Fragmentation analysis provisionally identified the kaempferol transglycosylation products as kaempferol 3-O, 7-O, and 4'-O glucosides and 3,7-O, 4',7-O, and 3,4'-O bis-O-glucosides. The Os9BGlu31 W243 mutants were also better able to use kaempferol 3-O-glucoside as a donor substrate.
  
  
  CONCLUSIONS
  The W243 residue was found to be critical to the substrate and product specificity of Os9BGlu31 transglucosidase and mutation of this residue allows production of a range of glucoconjugates.
authors: School of Biochemistry, Institute of Science, and Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors: Juthamath
authors: J
authors: Komvongsa
authors: School of Biochemistry, Institute of Science, and Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors: Sukanya
authors: S
authors: Luang
authors: Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA.
authors: Joaquim V
authors: JV
authors: Marques
authors: School of Biochemistry, Institute of Science, and Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors: Kannika
authors: K
authors: Phasai
authors: Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA.
authors: Laurence B
authors: LB
authors: Davin
authors: Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA.
authors: Norman G
authors: NG
authors: Lewis
authors: School of Biochemistry, Institute of Science, and Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.; Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok, Thailand. Electronic address: cairns@sut.ac.th.
authors: James R
authors: JR
authors: Ketudat Cairns
mesh_terms: D020134:Catalytic Domain / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D005959:Glucosidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D044949:Kaempferols / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D009005:Monosaccharides / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D012275:Oryza / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D044949:Kaempferols; D009005:Monosaccharides; D050505:Mutant Proteins; D010940:Plant Proteins; C511963:kaempferol-3-O-glucoside; D005959:Glucosidases
keywords: Electrospray ionization mass spectrometry; Glycoside hydrolase family GH1; Glycosylation; Rice (Oryza sativa L.); Site-directed mutagenesis; Transglucosidase