title: Molecular characterization and heterologous expression of a Xanthophyllomyces dendrorhous α-glucosidase with potential for prebiotics production.
abstract: Basidiomycetous yeast Xanthophyllomyces dendrorhous expresses an α-glucosidase with strong transglycosylation activity producing prebiotic sugars such as panose and an unusual tetrasaccharides mixture including α-(1-6) bonds as major products, which makes it of biotechnological interest. Initial analysis pointed to a homodimeric protein of 60 kDa subunit as responsible for this activity. In this study, the gene Xd-AlphaGlu was characterized. The 4131-bp-long gene is interrupted by 13 short introns and encodes a protein of 990 amino acids (Xd-AlphaGlu). The N-terminal sequence of the previously detected 60 kDa protein resides in this larger protein at residues 583-602. Functionality of the gene was proved in Saccharomyces cerevisiae, which produced a protein of about 130 kDa containing Xd-AlphaGlu sequences. All properties of the heterologously expressed protein, including thermal and pH profiles, activity on different substrates, and ability to produce prebiotic sugars were similar to that of the α-glucosidase produced in X. dendrorhous. No activity was detected in S. cerevisiae containing exclusively the 1256-bp from gene Xd-AlphaGlu that would encode synthesis of the 60 kDa protein previously detected. Data were compatible with an active monomeric α-glucosidase of 990 amino acids and an inactive hydrolysis product of 60 kDa. Protein Xd-AlphaGlu contained most of the elements characteristic of α-glucosidases included in the glycoside hydrolases family GH31 and its structural model based on the homologous human maltase-glucoamylase was obtained. Remarkably, the Xd-AlphaGlu C-terminal domain presents an unusually long 115-residue insertion that could be involved in this enzyme's activity against long-size substrates such as maltoheptaose and soluble starch.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM), Nicolás Cabrera 1. Universidad Autónoma Madrid, 28049, Madrid, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Gutiérrez-Alonso
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM), Nicolás Cabrera 1. Universidad Autónoma Madrid, 28049, Madrid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Gimeno-Pérez
authors.affiliation: Departamento de Cristalografía y Biología Estructural, Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006, Madrid, Spain.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Ramírez-Escudero
authors.affiliation: Instituto de Catálisis y Petroleoquímica, CSIC, Marie Curie 2, 28049, Madrid, Spain.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Plou
authors.affiliation: Departamento de Cristalografía y Biología Estructural, Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006, Madrid, Spain.
authors.forename: Julia
authors.initials: J
authors.lastname: Sanz-Aparicio
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM), Nicolás Cabrera 1. Universidad Autónoma Madrid, 28049, Madrid, Spain. mfernandez@cbm.csic.es.
authors.forename: María
authors.initials: M
authors.lastname: Fernández-Lobato
mesh_terms: D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000737:chemistry* / Q000201:enzymology; D003001:Cloning, Molecular; D005091:Exons; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005936:Glucans / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007438:Introns; D007700:Kinetics; D008958:Models, Molecular; D008970:Molecular Weight; D016366:Open Reading Frames; D010802:Phylogeny; D056692:Prebiotics; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013213:Starch / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D005936:Glucans; D056692:Prebiotics; D021122:Protein Subunits; D011994:Recombinant Proteins; C008763:panose; C031146:maltoheptaose; D013213:Starch; D000520:alpha-Glucosidases
keywords: Alpha-glucosidase; GH31 family; Maltooligosaccharides; Panose; Xanthophyllomyces dendrorhous