title: Mutagenesis of key amino acids alters activity of a Saccharomyces cerevisiae endo-polygalacturonase expressed in Pichia pastoris.
abstract: A polygalacturonase (PG)-encoding gene from Saccharomyces cerevisiae (PGU1) was successfully expressed in the methylotrophic yeast Pichia pastoris. PG secretion was efficiently directed by the S. cerevisiae alpha-factor signal sequence, while the native (PGU1) leader peptide was unable to direct protein export in P. pastoris. The level of PGU1 activity achieved in P. pastoris was significantly enhanced when compared to activity using the same gene in S. cerevisiae. Expression of PG proteins, engineered by site-directed mutagenesis, in P. pastoris showed that aspartic acid residues at positions 179, 200, and 201, and histidine 222 were essential for enzyme activity. Mutation of the two potential glycosylation sites in PGU1 showed that the two residues individually (N318D, N330D) did not affect secreted enzyme activity, but the double mutant caused a 50% reduction in enzyme activity when compared to the wild-type PGU1 transformant.
authors.affiliation: Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK.
authors.forename: Pilar
authors.initials: P
authors.lastname: Blanco
authors.forename: Graham
authors.initials: G
authors.lastname: Thow
authors.forename: Craig G
authors.initials: CG
authors.lastname: Simpson
authors.forename: Tomas G
authors.initials: TG
authors.lastname: Villa
authors.forename: Brian
authors.initials: B
authors.lastname: Williamson
mesh_terms: D019943:Amino Acid Substitution; D005822:Genetic Vectors / Q000235:genetics; D000432:Methanol; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics*; D011096:Polygalacturonase / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D011994:Recombinant Proteins; D011096:Polygalacturonase; D000432:Methanol