title: Extra EF hand unit (DX) mediated stabilization and calcium independency of α-amylase.
abstract: It is the common feature of α-amylases that calcium ion is required for their structural integrity and thermal stability. All amylases have at least one Ca(2+) per molecule; therefore amino acids involved in calcium binding are specific and conserved. In this study, sequence analysis revealed the presence of EF-hand-like motif in calcium-binding loop of Bacillus megaterium WHO (BMW)-amylase that was previously isolated from BMW. The EF-hand motif and its variants (EF-hand-like motif) are the most common calcium-binding motifs found in a large number of protein families. To investigate the effect of calcium ion on the thermal stability and activity of BMW-amylase, we used site-directed mutagenesis to replace histidine 58 with Asp (D), Ile (I), Tyr (Y), Phe (F), and Arg (R) at the seventh position of EF-hand-like motif. Upon the addition of an extra DX unit to the calcium-binding loop in H58D variant, thermal stability, catalytic activity, and chelating power of the enzyme improved due to higher affinity toward calcium. H58D variant demonstrated calcium independency compared to the wild type and other created mutants. Conformational changes in the presence and absence of Ca(2+) were monitored using fluorescence technique.
authors.affiliation: Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran.
authors.forename: Leila
authors.initials: L
authors.lastname: Sadeghi
authors.forename: Khosro
authors.initials: K
authors.lastname: Khajeh
authors.forename: Nasrin
authors.initials: N
authors.lastname: Mollania
authors.forename: Bahareh
authors.initials: B
authors.lastname: Dabirmanesh
authors.forename: Bijan
authors.initials: B
authors.lastname: Ranjbar
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D020832:EF Hand Motifs / Q000235:genetics*; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D058849:Protein Refolding; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000516:alpha-Amylases; D002118:Calcium