Determination of the second autoproteolytic cleavage site of cephalosporin C acylase and the effect of deleting its flanking residues in the α-C-terminal region

• The second cleavage site of CPC acylase was identified as A232–S233 by α-C-terminus tandem MS/MS.
• The spacer peptide released after two-step auto-cleavages was 7 aa in length.
• Importance of the cleavage-site-flanking residues in the α-C-terminal region was revealed.
• Residue G229 was crucial for the first cleavage of Pre-sCPCAcy.
• Mutant protein D4 with 212-ADLA-215 deletion formally activated with 102%-enhanced Kcat/Km.

Self-activation through two-step intra-molecular cleavages is of great importance for the synthesis of mature and functional cephalosporin acylase in the N-terminal nucleophile (Ntn) hydrolase superfamily. A synthetic gene for cephalosporin C acylase (sCPCAcy) from Pseudomonas sp. SE83 was overexpressed, and the self-activated sCPCAcy was produced in Escherichia coli JM109(DE3)/pET28-sCPCAcy. The first autoproteolytic cleavage site of Pre-sCPCAcy was determined to be G239–S240 according to the common features of Ntn hydrolases. The second cleavage site was identified as A232–S233 by C-terminus tandem MS/MS analysis of the purified α-subunit, which released a 7-aa spacer peptide with the generation of the α and β subunits of the mature sCPCAcy. The effect of the cleavage-site-flanking residues in the α-C-terminal region of sCPCAcy on its activation and characteristics was further evaluated. Residue G229 was found to be crucial for the first cleavage of Pre-sCPCAcy. Deletions in the α-C-terminal region were performed, and 14 mutant proteins were constructed. The majority of the fragment-deleted mutant proteins completely lost their activity due to failure of the first autocleavage, but this loss was not observed in mutant proteins D2 (227-AM-228 deletion) and D4 (212-ADLA-215 deletion), which formally activated into mature sCPCAcy with high activity. The Kcat/Km values of mutant proteins D2 and D4 were 46% and 102% higher than that of the original control, respectively.