title: Glycosylation variants of a β-glucosidase secreted by a Taiwanese fungus, Chaetomella raphigera, exhibit variant-specific catalytic and biochemical properties.
abstract: Cellulosic biomass is an abundant and promising energy source. To make cellulosic biofuels competitive against conventional fuels, conversion of rigid plant materials into sugars must become efficient and cost-effective. During cellulose degradation, cellulolytic enzymes generate cellobiose (β-(1→4)-glucose dimer) molecules, which in turn inhibit such enzymes by negative feedback. β-Glucosidases (BGLs) cleave cellobiose into glucose monomers, assisting overall cellulolytic activities. Therefore, BGLs are essential for efficient conversion of cellulosic biomass into biofuels, and it is important to characterize newly isolated BGLs for useful traits. Here, we report our discovery that the indigenous Taiwanese fungus Chaetomella raphigera strain D2 produces two molecular weight variants of a single BGL, D2-BGL (shortened to "D2"), which differ in O-glycosylation. The more extensively O-glycosylated form of native D2 (nD2L) has increased activity toward the natural substrate, cellobiose, compared to the less O-glycosylated form (nD2S). nD2L is more stable at 60°C, in acidic pH, and in the presence of the ionic detergent sodium dodecyl sulfate than nD2S. Furthermore, unlike nD2S, nD2L does not display substrate inhibition by an artificial substrate p-nitrophenyl glucopyranoside (pNPG), and the glucose feedback inhibition kinetics of nD2L is competitive (while it is non-competitive for nD2S), suggesting that these two glycovariants of D2 bind substrates differently. Interestingly, D2 produced in a heterologous system, Pichia pastoris, closely mimics properties of nD2S. Our studies suggest that O-glycosylation of D2 is important in determining its catalytic and biochemical properties.
authors.affiliation: Department of Biology, Washington University in St. Louis, St. Louis, Missouri, United States of America; Department of Pathology and Immunology, School of Medicine, Washington University in St. Louis, St. Louis, Missouri, United States of America.
authors.forename: Aki
authors.initials: A
authors.lastname: Yoneda
authors.affiliation: Department of Environmental Science and Engineering, Tunghai University, Taichung, Taiwan, Republic of China.
authors.forename: Hsion-Wen David
authors.initials: HW
authors.lastname: Kuo
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, United States of America.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Ishihara
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, United States of America.
authors.forename: Parastoo
authors.initials: P
authors.lastname: Azadi
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China; Agricultural Biotechnology Center, National Chung-Hsing University, Taichung, Taiwan, Republic of China; Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan, Republic of China.
authors.forename: Su-May
authors.initials: SM
authors.lastname: Yu
authors.affiliation: Department of Biology, Washington University in St. Louis, St. Louis, Missouri, United States of America; Agricultural Biotechnology Center, National Chung-Hsing University, Taichung, Taiwan, Republic of China; Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan, Republic of China; Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China.
authors.forename: Tuan-hua David
authors.initials: TH
authors.lastname: Ho
mesh_terms: D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D055162:Biocatalysis / Q000187:drug effects; D002475:Cellobiose / Q000378:metabolism; D002850:Chromatography, Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D005947:Glucose / Q000494:pharmacology; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity / Q000187:drug effects; D013624:Taiwan; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D007527:Isoenzymes; D011134:Polysaccharides; D002475:Cellobiose; D012967:Sodium Dodecyl Sulfate; D001617:beta-Glucosidase; D005947:Glucose