Download MendeleyStructural Basis of Increased Resistance to Thermal Denaturation Induced by Single Amino Acid Substitution in the Sequence of Beta-Glucosidase a from Bacillus Polymyxa.
Sanz-Aparicio, J., Hermoso, J.A., Martinez-Ripoll, M., Gonzalez, B., Lopez-Camacho, C., Polaina, J.(1998) Proteins 33: 567-576
- PubMed: 9849940
- DOI: https://doi.org/10.1002/(sici)1097-0134(19981201)33:4<567::aid-prot9>3.0.co;2-u
- Primary Citation of Related Structures:
1E4I - PubMed Abstract:
The increasing development of the biotechnology industry demands the design of enzymes suitable to be used in conditions that often require broad resistance against adverse conditions. beta-glucosidase A from Bacillus polymyxa is an interesting model for studies of protein engineering. This is a well-characterized enzyme, belonging to glycosyl hydrolase family 1. Its natural substrate is cellobiose, but is also active against various artificial substrates. In its native state has an octameric structure. Its subunit conserves the general (alpha/beta)8 barrel topology of its family, with the active site being in a cavity defined along the axis of the barrel. Using random-mutagenesis, we have identified several mutations enhancing its stability and it was found that one them, the E96K substitution, involved structural changes. The crystal structure of this mutant has been determined by X-ray diffraction and compared with the native structure. The only difference founded between both structures is a new ion pair linking Lys96 introduced at the N-terminus of helix alpha2, to Asp28, located in one of the loops surrounding the active-site cavity. The new ion pair binds two segments of the chain that are distant in sequence and, therefore, this favorable interaction must exert a determinant influence in stabilizing the tertiary structure. Furthermore, analysis of the crystallographic isotropic temperature factors reveals that, as a direct consequence of the introduced ion pair, an unexpected decreased mobility of secondary structure units of the barrel which are proximal to the site of mutation is observed. However, this effect is observed only in the surrounding of one of the partners forming the salt bridge and not around the other. These results show that far-reaching effects can be achieved by a single amino acid replacement within the protein structure. Consequently, the identification and combination of a few single substitutions affecting stability may be sufficient to obtain a highly resistant enzyme, suitable to be used under extreme conditions.
Organizational Affiliation:
Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química-Física Rocasolano, CSIC, Madrid, Spain. [email protected]
