X‐ray crystallographic and mutational analysis of the NylC precursor: catalytic mechanism of autocleavage and substrate hydrolysis of nylon hydrolase

Nylon hydrolase (NylC), a member of the N‐terminal nucleophile (Ntn) hydrolase superfamily, is responsible for the degradation of various aliphatic nylons, including nylon‐6 and nylon‐66. NylC is initially expressed as an inactive precursor (36 kDa), but the precursor is autocatalytically cleaved at Asn266/Thr267 to generate an active enzyme composed of 27 and 9 kDa subunits. We isolated various mutants with amino acid changes at the catalytic centre. X‐ray crystallographic analysis revealed that the NylC precursor forms a doughnut‐shaped quaternary structure composed of four monomers (molecules A‐D) with D2 symmetry. Catalytic residues in the precursor are covered by loop regions at the A/B interface (equivalent to the C/D interface). However, the catalytic residues are exposed to the solvent environment through autocleavage followed by movements of the loop regions. T267A, D306A and D308A mutations resulted in a complete loss of autocleavage. By contrast, in the T267S mutant, autocleavage proceeded slowly at a constant reaction rate (k = 2.8 × 10−5 s−1) until complete conversion, but the reaction was inhibited by K189A and N219A mutations. Based on the crystallographic and molecular dynamic simulation analyses, we concluded that the Asp308‐Asp306‐Thr267 triad, resembling the Glu‐Ser‐Ser triad conserved in Ntn‐hydrolase family enzymes, is responsible for autocleavage and that hydrogen‐bonding networks connecting Thr267 with Lys189 and Asn219 are required for increasing the nucleophilicity of Thr267‐OH in both the water accessible and water inaccessible systems. Furthermore, we determined that NylC employs the Asp308‐Asp306‐Thr267 triad as catalytic residues for substrate hydrolysis, but the reaction requires Lys189 and Tyr146 as additional catalytic/substrate‐binding residues specific for nylon hydrolysis. Nylon hydrolase (NylC), responsible for the degradation of aliphatic nylons, is initially expressed as an inactive precursor. We introduced various mutations to the catalytic residue Thr267 and its surrounding residues by site‐directed mutagenesis and examined the effects on autoprocessing. We determined the three‐dimensional structure of the precursor, analysed the structural alterations induced by autocleavage and determined the precise catalytic mechanisms of autocleavage and substrate hydrolysis.