Structural Investigation and Homology Modeling Studies of Native and Truncated Forms of α-Amylases from Sclerotinia sclerotiorum
2009
Ben Abdelmalek, Imen, INSAT, Centre Urbain Nord, Tunis Cedex, Tunisia | Urdaci, Maria Camino, LMBA, Pessac Cedex, France | Ali, Mamdouh Ben, Centre de Biotechnologie de Sfax, Sfax, Tunisia | Denayrolles, Muriel, LMBA, Pessac Cedex, France | Chaignepain, Stephane, Institut Europeen de Chimie et Biologie (IECB), Pessac Cedex, France | Limam, Ferid, INSAT, Centre Urbain Nord, Tunis Cedex, Tunisia | Bejar, Samir, Centre de Biotechnologie de Sfax, Sfax, Tunisia | Marzouki, Mohamed Nejib, INSAT, Centre Urbain Nord, Tunis Cedex, Tunisia
The filamentous ascomycete Sclerotinia sclerotiorum is well known for its ability to produce a large variety of hydrolytic enzymes. Two α-amylases ScAmy54 and ScAmy43 predicted to play an important role in starch degradation were showed to produce specific oligosaccharides essentially maltotriose that have a considerable commercial interest. Primary structure of the two enzymes was established by N-terminal sequencing, MALDI-TOF masse spectrometry and cDNA cloning. The two proteins have the same N-terminal catalytic domain and ScAmy43 derived from ScAmy54 by truncation of 96 amino acids at the carboxyl-terminal region. Data of genomic analysis suggested that the two enzymes originated from the same α-amylase gene and that truncation of ScAmy54 to ScAmy43 occurred probably during 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 residues. ScAmy54 exhibited high amino acid homology with other liquefying fungal α-amylases essentially in the four conserved regions and in the putative catalytic triad. A 3-D structure model of ScAmy54 and ScAmy43 was built using the 3-D structure of 2guy from A. niger as template. ScAmy54 is composed by three domains A, B, and C, including the well-known (β/α)∧8 barrel motif in domain A, have a typical structure of α-amylase family, whereas ScAmy43 contained only tow domains A and B is the first fungal α-amylase described until now with the smallest catalytic domain.
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