title: Structural and functional insights into Saccharomyces cerevisiae riboflavin biosynthesis reductase RIB7.
abstract: Saccharomyces cerevisiae RIB7 (ScRIB7) is a potent target for anti-fungal agents because of its involvement in the riboflavin biosynthesis pathway as a NADPH-dependent reductase. However, the catalytic mechanism of riboflavin biosynthesis reductase (RBSRs) is controversial, and enzyme structure information is still lacking in eukaryotes. Here we report the crystal structure of Saccharomyces cerevisiae RIB7 at 2.10 Å resolution and its complex with NADPH at 2.35 Å resolution. ScRIB7 exists as a stable homodimer, and each subunit consists of nine central β-sheets flanked by five helices, resembling the structure of RIB7 homologues. A conserved G(76)-X-G(78)-Xn-G(181)-G(182) motif is present at the NADPH pyrophosphate group binding site. Activity assays confirmed the necessity of Thr79, Asp83, Glu180 and Gly182 for the activity of ScRIB7. Substrate preference of ScRIB7 was altered by mutating one residue (Thr35) to a Lysine, implying that ScRIB7 Thr35 and its corresponding residue, a lysine in bacteria, are important in substrate-specific recognition.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Zongyang
authors.initials: Z
authors.lastname: Lv
authors.forename: Jian
authors.initials: J
authors.lastname: Sun
authors.forename: Yingfang
authors.initials: Y
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002151:Calorimetry; D003067:Coenzymes; D018360:Crystallography, X-Ray; D057075:Enzyme Assays; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D012256:Riboflavin / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D003067:Coenzymes; D029701:Saccharomyces cerevisiae Proteins; D009249:NADP; D010088:Oxidoreductases; C584195:RIB7 protein, S cerevisiae; D012256:Riboflavin