title: Mutational analysis of Kex2 recognition sites and a disulfide bond in tannase from Aspergillus oryzae.
abstract: Aspergillus oryzae tannase (AoTanA), which contains two Kex2 recognition sites at positions Arg311 and Arg316, consists of two subunits that are generated by the cleavage of tannase gene product by the Kex2 protease. Based on the crystal structure of feruloyl esterase from Aspergillus oryzae (AoFaeB), which has been classified as a member of the fungal tannase family, the catalytic triad residues of AoTanA are predicted to be Ser195, Asp455, and His501, with the serine and histidine residues brought together by a disulfide bond of the neighboring cysteines, Cys194 and Cys502. In this study, we investigated the functional role of the Kex2 recognition sites and disulfide bond between the neighboring cysteines in AoTanA. We constructed a double variant (R311A/R316A), a seven amino-acid deletion variant of region Lys310-Arg316 (ΔKR), and two single variants (C194A and C502A). While the R311A/R316A variant exhibited the two bands similar to wild type by SDS-PAGE after treatment with endoglycosidase H, the ΔKR variant exhibited only one band. R311A/R316A variation had no effect on tannase activity and stability. Meanwhile, the ΔKR variant exhibited higher activity compared to the wild-type. The activities of the C194A and C502A variants decreased considerably (<0.24% of the wild-type) toward methyl gallate.
authors.affiliation: Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka, 997-8555, Japan. Electronic address: tkoseki@tds1.tr.yamagata-u.ac.jp.
authors.forename: Takuya
authors.initials: T
authors.lastname: Koseki
authors.affiliation: Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka, 997-8555, Japan.
authors.forename: Motohiro
authors.initials: M
authors.lastname: Otsuka
authors.affiliation: Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka, 997-8555, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Mizuno
authors.affiliation: Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka, 997-8555, Japan.
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Shiono
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology*; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry*; D002384:Catalysis; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D004220:Disulfides / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry*; D017353:Gene Deletion; D014644:Genetic Variation; D006026:Glycoside Hydrolases / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D043484:Proprotein Convertases / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004220:Disulfides; D005656:Fungal Proteins; D012694:Serine; D006639:Histidine; D002265:Carboxylic Ester Hydrolases; C022711:tannase; C070196:feruloyl esterase; D006026:Glycoside Hydrolases; D043484:Proprotein Convertases; D003545:Cysteine
keywords: Aspergillus oryzae; Disulfide bond; Kex2 recognition site; Tannase