title: Structures of alkaloid biosynthetic glucosidases decode substrate specificity.
abstract: Two similar enzymes with different biosynthetic function in one species have evolved to catalyze two distinct reactions. X-ray structures of both enzymes help reveal their most important differences. The Rauvolfia alkaloid biosynthetic network harbors two O-glucosidases: raucaffricine glucosidase (RG), which hydrolyses raucaffricine to an intermediate downstream in the ajmaline pathway, and strictosidine glucosidase (SG), which operates upstream. RG converts strictosidine, the substrate of SG, but SG does not accept raucaffricine. Now elucidation of crystal structures of RG, inactive RG-E186Q mutant, and its complexes with ligands dihydro-raucaffricine and secologanin reveals that it is the "wider gate" of RG that allows strictosidine to enter the catalytic site, whereas the "slot-like" entrance of SG prohibits access by raucaffricine. Trp392 in RG and Trp388 in SG control the gate shape and acceptance of substrates. Ser390 directs the conformation of Trp392. 3D structures, supported by site-directed mutations and kinetic data of RG and SG, provide a structural and catalytic explanation of substrate specificity and deeper insights into O-glucosidase chemistry.
authors: Institute of Materia Medica, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, PR China.
authors: Liqun
authors: L
authors: Xia
authors: Martin
authors: M
authors: Ruppert
authors: Meitian
authors: M
authors: Wang
authors: Santosh
authors: S
authors: Panjikar
authors: Haili
authors: H
authors: Lin
authors: Chitra
authors: C
authors: Rajendran
authors: Leif
authors: L
authors: Barleben
authors: Joachim
authors: J
authors: Stöckigt
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019143:Evolution, Molecular; D005959:Glucosidases / Q000737:chemistry / Q000378:metabolism*; D057888:Iridoid Glucosides / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011926:Rauwolfia / Q000737:chemistry / Q000201:enzymology*; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism; D014748:Vinca Alkaloids / Q000378:metabolism*
chemical_list: D057888:Iridoid Glucosides; D010940:Plant Proteins; D014748:Vinca Alkaloids; C021086:secologanin; C021093:strictosidine; D012694:Serine; D014364:Tryptophan; D005959:Glucosidases; C119910:raucaffricine beta-glucosidase; C456847:strictosidine glucosidase