Alteration of substrate selection of antibiotic acylase from β-lactam to echinocandin.

The antibiotic acylases belonging to the N-terminal nucleophile hydrolase superfamily are key enzymes for the industrial production of antibiotic drugs. Cephalosporin acylase (CA) and penicillin G acylase (PGA) are two of the most intensively studied enzymes that catalyze the deacylation of β-lactam antibiotics. On the other hand, aculeacin A acylase (AAC) is known to be an alternative acylase class catalyzing the deacylation of echinocandin or cyclic lipopeptide antibiotic compounds, but its structural and enzymatic properties remain to be explored. In the present study, 3D homology models of AAC were constructed, and docking simulation with substrate ligands was performed for AAC, as well as for CA and PGA. The docking models of AAC with aculeacin A suggest that AAC has the deep narrow binding pocket for the long-chain fatty acyl group of the echinocandin molecule. To confirm this, CA mutants have been designed to form the binding pocket for the long acyl chain. Experimentally synthesized mutant enzymes exhibited lower enzymatic activity for cephalosporin but higher activity for aculeacin A, in comparison with the wild-type enzyme. The present results have clarified the difference in mechanisms of substrate selection between the β-lactam and echinocandin acylases and demonstrate the usefulness of the computational approaches for engineering the enzymatic properties of antibiotic acylases.

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