Computer-aided subsite mapping of α-amylases

Subsite mapping is a crucial procedure in the characterization of α-amylases (EC 3.2.1.1), which are extensively used in starch-based industries and in diagnosis of pancreatic and salivary glands disorders. A computer-aided method has been developed for subsite mapping of α-amylases, which substitutes the difficult, expensive, and time-consuming experimental determination of action patterns to crystal structures based energy calculations. Interaction energies between enzymes and carbohydrate substrates were calculated after short energy minimization by a molecular mechanics program. A training set of wild type and mutant amylases with known experimental action patterns of 13 enzymes of wide range of origin was used to set up the procedure. Calculations for training set resulted in good correlation in case of subsite binding energies (r2 = 0.827–0.929) and bond cleavage frequencies (r2 = 0.727–0.835). A set of eight novel barley amylase 1 mutants was used to test our model. Subsite binding energies were predicted with r2 = 0.502 correlation coefficient, while bond cleavage frequency prediction resulted in r2 = 0.538. Our computer-aided procedure may supplement the experimental subsite mapping methods to predict and understand characteristic features of α-amylases.

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► Subsite binding energies of homologous models of wild-type and mutant α-amylases were calculated by a molecular mechanical program.
► Parameters of the calculation were set up to get correlation between the calculated and the experimental binding energies of enzymes of a training set.
► Calculations on an independent test set also resulted in good correlation, except mutants containing neutral to charged residue substitution.
► Our computer-aided procedure may help to understand structure-function relationship of α-amylases and to design enzymes with new features.