title: New insight into the catalytic properties of rice sucrose synthase.
abstract: Sucrose synthase (SuS), which catalyzes the reversible conversion of sucrose and uridine diphosphate (UDP) into fructose and UDP-glucose, is a key enzyme in sucrose metabolism in higher plants. SuS belongs to family 4 of the glycosyltransferases (GT4) and contains an E-X7-E motif that is conserved in members of GT4 and two other GT families. To gain insight into the roles of this motif in rice sucrose synthase 3 (RSuS3), the two conserved glutamate residues (E678 and E686) in this motif and a phenylalanine residue (F680) that resides between the two glutamate residues were changed by site-directed mutagenesis. All mutant proteins maintained their tetrameric conformation. The mutants E686D and F680Y retained partial enzymatic activity and the mutants E678D, E678Q, F680S, and E686Q were inactive. Substrate binding assays indicated that UDP and fructose, respectively, were the leading substrates in the sucrose degradation and synthesis reactions of RSuS3. Mutations on E678, F680, and E686 affected the binding of fructose, but not of UDP. The results indicated that E678, F680, and E686 in the E-X7-E motif of RSuS3 are essential for the activity of the enzyme and the sequential binding of substrates. The sequential binding of the substrates implied that the reaction catalyzed by RSuS can be controlled by the availability of fructose and UDP, depending on the metabolic status of a tissue.
authors.affiliation: Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617, Taiwan.
authors.forename: Yu-Chiao
authors.initials: YC
authors.lastname: Huang
authors.affiliation: Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617, Taiwan.
authors.forename: Erh-Chieh
authors.initials: EC
authors.lastname: Hsiang
authors.affiliation: Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617, Taiwan. ccyang@ntu.edu.tw.
authors.forename: Chien-Chih
authors.initials: CC
authors.lastname: Yang
authors.affiliation: Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617, Taiwan. aywang@ntu.edu.tw.
authors.forename: Ai-Yu
authors.initials: AY
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D055162:Biocatalysis; D005632:Fructose / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D016415:Sequence Alignment; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism*; D014530:Uridine Diphosphate / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D005632:Fructose; D013395:Sucrose; D014530:Uridine Diphosphate; D005964:Glucosyltransferases; C022699:sucrose synthase; D014532:Uridine Diphosphate Glucose
keywords: Enzyme kinetics; Molecular modeling; Site-directed mutagenesis; Substrate binding; Sucrose synthase