title: An amino acid at position 512 in β-glucosidase from Clavibacter michiganensis determines the regioselectivity for hydrolyzing gypenoside XVII.
abstract: A recombinant β-glucosidase from Clavibacter michiganensis specifically hydrolyzed the outer and inner glucose linked to the C-3 position in protopanaxadiol (PPD)-type ginsenosides and the C-6 position in protopanaxatriol (PPT)-type ginsenosides except for the hydrolysis of gypenoside LXXV (GypLXXV). The enzyme converted gypenoside XVII (GypXVII) to GypLXXV by hydrolyzing the inner glucose linked to the C-3 position. The substrate-binding residues obtained from the GypXVII-docked homology models of β-glucosidase from C. michiganensis were replaced with alanine, and the amino acid residue at position 512 was selected because of the changed regioselectivity of W512A. Site-directed mutagenesis for the amino acid residue at position 512 was performed. W512A and W512K hydrolyzed the inner glucose linked to the C-3 position and the outer glucose linked to the C-20 position of GypXVII to produce GypLXXV and F2. W512R hydrolyzed only the outer glucose linked to the C-20 position of GypXVII to produce F2. However, W512E and W512D exhibited no activity for GypXVII. Thus, the amino acid at position 512 is a critical residue to determine the regioselectivity for the hydrolysis of GypXVII. These wild-type and variant enzymes produced diverse ginsenosides, including GypXVII, GypLXXV, F2, and compound K, from ginsenoside Rb1. To the best of our knowledge, this is the first report of the alteration of regioselectivity on ginsenoside hydrolysis by protein engineering.
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul, 143-701, Republic of Korea.
authors.forename: Kyung-Chul
authors.initials: KC
authors.lastname: Shin
authors.forename: Seung-Hye
authors.initials: SH
authors.lastname: Hong
authors.forename: Min-Ju
authors.initials: MJ
authors.lastname: Seo
authors.forename: Deok-Kun
authors.initials: DK
authors.lastname: Oh
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029774:Gynostemma / Q000737:chemistry / Q000378:metabolism; D008835:Micrococcaceae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D010936:Plant Extracts / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D010936:Plant Extracts; C474918:gypenoside; D001617:beta-Glucosidase