ABSTRACT
In the Bacillus amyloliquefaciens α-amylase (BAA), the loop (residues 176-185; region I) that is the part of the calcium-binding site (CaI, II) has two more amino acid residues than the α-amylase from Bacillus licheniformis (BLA). Arg176 in this region makes an ionic interaction with Glu126 from region II (residues 118-130), but this interaction is lost in BLA owing to substitution of R176Q and E126V. The goal of the present work was to quantitatively estimate the effect of ionic interaction on the overall stability of the enzyme. To clarify the functional and structural significance of the corresponding salt bridge, Glu126 was deleted (ΔE126) and converted to Val (E126V), Asp (E126D), and Lys (E126K) by site-directed mutagenesis. Kinetic constants, thermodynamic parameters, and structural changes were examined for the wild-type and mutated forms using UV-visible, atomic absoption, and fluorescence emission spectroscopies. Wild type exhibited higher k(cat) and K(m) but lower catalytic efficiency than the mutant enzymes. A decreased thermostability and an increased flexibility were also found in all of the mutant enzymes when compared with the wild type. Additionally, the calcium content of the wild type was more than ΔE126. Thus, it may be suggested that ionic interaction could decrease the mobility of the discussed region, prevent the diffusion of cations, and improve the thermostability of the whole enzyme. Based on these observations, the contribution of loop destabilization may be compensated by the formation of a salt bridge that has been used as an evolutionary mechanism or structural adaptation by the mesophilic enzyme.
Subject(s)
Bacillus/enzymology , alpha-Amylases/chemistry , alpha-Amylases/metabolism , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Calcium/metabolism , DNA Mutational Analysis , Enzyme Stability , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Binding , Protein Conformation , Protein Stability , Spectrum Analysis , Thermodynamics , alpha-Amylases/geneticsABSTRACT
Nerve tissue engineering requires suitable precursor cells as well as the necessary biochemical and physical cues to guide neurite extension and tissue development. An ideal scaffold for neural regeneration would be both fibrous and electrically conductive. We have contrasted the growth and neural differentiation of mouse embryonic stem cells on three different aligned nanofiber scaffolds composed of poly L: -lactic acid supplemented with either single- or multi-walled carbon-nanotubes. The addition of the nanotubes conferred conductivity to the nanofibers and promoted mESC neural differentiation as evidenced by an increased mature neuronal markers expression. We propose that the conductive scaffold could be a useful tool for the generation of neural tissue mimics in vitro and potentially as a scaffold for the repair of neural defects in vivo.
