title: Identification of a new oat β-amylase by functional proteomics.
abstract: Oat (Avena sativa L.) seed extracts exhibited a high degree of catalytic activity including amylase activities. Proteins in the oat seed extracts were optimized for their amylolytic activities. Oat extract with amylolytic activity was separated by SDS-PAGE and a major protein band with an apparent molecular mass of 53 kDa was subjected to tryptic digestion. The generated amino acid sequences were analyzed by liquid chromatography–tandem mass spectrometry (LC/ESI/MS/MS) and database searches. These sequences were used to identify a partial cDNA from expressed sequence tags (ESTs) of A. sativa L. Based upon EST sequences, a predicted full-length gene was identified, with an open reading frame of 1464 bp encoding a protein of 488 amino acid residues (AsBAMY), with a theoretical molecular mass of 55 kDa identified as a β-amylase belonging to the plant β-amylase family. Primary structure of oat β-amylase (AsBAMY) protein indicated high similarity with other β-amylase from other cereals such as wheat (Triticum aestivum), barley (Hordeum vulgare), and rye (Secale cereale) with two conserved Glu residues (E184 and E378) assigned as the “putative” catalytic residues which would act as an acid and base pair in the catalytic process. In addition, a 3D-model of AsBAMY was built from known X-ray structures and sequence alignments. A similar core (β/α)8-barrel architecture was found in AsBAMY like the other cereal β-amylases with a specific location of the active site in a pocket-like cavity structure made at one end of this core (β/α)8-barrel domain suggesting an accessibility of the non-reducing end of the substrate and thus confirming the results of AsBAMY exo-acting hydrolase.
authors.affiliation: Biological Engineering Department, Ecole nationale d'Ingénieurs de Sfax, University of Sfax, Tunisia.
authors.forename: Nihed
authors.initials: N
authors.lastname: Ben Halima
authors.affiliation: Laboratory of Plant Biotechnology, Faculté des Sciences de Sfax, University of Sfax, Tunisia.
authors.forename: Bassem
authors.initials: B
authors.lastname: Khemakhem
authors.affiliation: Laboratory of Plant Biotechnology, Faculté des Sciences de Sfax, University of Sfax, Tunisia.
authors.forename: Imen
authors.initials: I
authors.lastname: Fendri
authors.affiliation: Bioinformatics Center, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Ogata
authors.affiliation: Centre de Biophysique moléculaire, CNRS UPR4301 and Université d'Orléans, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Baril
authors.affiliation: Centre de Biophysique moléculaire, CNRS UPR4301 and Université d'Orléans, France.
authors.forename: Chantal
authors.initials: C
authors.lastname: Pichon
authors.affiliation: Biological Engineering Department, Ecole nationale d'Ingénieurs de Sfax, University of Sfax, Tunisia. Electronic address: slim.abdelkafi@enis.rnu.tn.
authors.forename: Slim
authors.initials: S
authors.lastname: Abdelkafi
mesh_terms: D000595:Amino Acid Sequence; D018554:Avena / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002853:Chromatography, Liquid; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D012639:Seeds / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry; D001614:beta-Amylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D010940:Plant Proteins; D001614:beta-Amylase
keywords: Avena sativa L.; Bioinformatic tools; Mass spectrometry; β-Amylase