title: Structural investigation and homology modeling studies of native and truncated forms of alpha-amylases from Sclerotinia sclerotiorum.
abstract: The filamentous ascomycete Sclerotinia sclerotiorum is well known for its ability to produce a large variety of hydrolytic enzymes for the degradation of plant polysaccharide material. Two alpha-amylases designated as ScAmy54 and ScAmy43 were biochemically characterized and predicted to play an important role in starch degradation. Those enzymes produce specific oligosaccharides, essentially maltotriose, that have a considerable commercial interest. The primary structures of the two enzymes were analyzed by N-terminal sequencing, MALDI-TOF mass spectrometry, and cDNA cloning, and implied that the two proteins have the same N-terminal catalytic domain and ScAmy43 was produced from ScAmy54 by truncation of 96 amino acids at the carboxyl-terminal region. The result of genomic analysis suggested that the two enzymes originated from the same alpha-amylase gene and that truncation of ScAmy54 to ScAmy43 occurred probably during the S. sclerotiorum cultivation. The structural gene of ScAmy54 consisted of 9 exons and 8 introns, containing a single 1,500-bp open reading frame encoding 499 amino acids including a signal peptide of 21 amino acids. ScAmy54 exhibited high amino acid identity to other liquefying fungal alpha-amylases, essentially in the four conserved regions and in the putative catalytic triad. A 3D structure model of ScAmy54 and ScAmy43 was built using the 3D structure of 2guy from A. niger as template. ScAmy54 with three domains A, B, and C, including the well-known (beta/alpha)8-barrel motif in domain A, has a typical structure of the alpha-amylase family. ScAmy43 composed only of domains A and B constitutes a smallest fungal alpha-amylase with only a catalytic domain.
authors.affiliation: Institut National des Sciences Appliquées et de Technologie, Centre Urbain Nord 676-1080 Tunis Cedex, Tunisia.
authors.forename: Imen
authors.initials: I
authors.lastname: Ben Abdelmalek
authors.forename: Maria Camino
authors.initials: MC
authors.lastname: Urdaci
authors.forename: Mamdouh
authors.initials: M
authors.lastname: Ben Ali
authors.forename: Muriel
authors.initials: M
authors.lastname: Denayrolles
authors.forename: Stephane
authors.initials: S
authors.lastname: Chaignepain
authors.forename: Ferid
authors.initials: F
authors.lastname: Limam
authors.forename: Samir
authors.initials: S
authors.lastname: Bejar
authors.forename: Mohamed Nejib
authors.initials: MN
authors.lastname: Marzouki
mesh_terms: D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics; D016681:Genome, Fungal; D008969:Molecular Sequence Data; D011485:Protein Binding; D021382:Protein Sorting Signals; D016415:Sequence Alignment; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics
chemical_list: D004271:DNA, Fungal; D021382:Protein Sorting Signals; D000516:alpha-Amylases; D002118:Calcium