The N-terminal Nucleophile Serine of Cephalosporin Acylase Executes the Second Autoproteolytic Cleavage and Acylpeptide Hydrolysis

Abstract

Cephalosporin acylase (CA) precursor is translated as a single polypeptide chain and folds into a self-activating pre-protein. Activation requires two peptide bond cleavages that excise an internal spacer to form the mature ␣␤ heterodimer. Using Q-TOF LC-MS, we located the second cleavage site between Glu 159 and Gly 160 , and detected the corresponding 10-aa spacer 160 GDPPDLADQG 169 of CA mutants. The site of the second cleavage depended on Glu 159 : moving Glu into the spacer or removing 5-10 residues from the spacer sequence resulted in shorter spacers with the cleavage at the carboxylic side of Glu. The mutant E159D was cleaved more slowly than the wild-type, as were mutants G160A and G160L. This allowed kinetic measurements showing that the second cleavage reaction was a firstorder, intra-molecular process. Glutaryl-7-aminocephalosporanic acid is the classic substrate of CA, in which the N-terminal Ser 170 of the ␤-subunit, is the nucleophile. Glu and Asp resemble glutaryl, suggesting that CA might also remove N-terminal Glu or Asp from peptides. This was indeed the case, suggesting that the N-terminal nucleophile also performed the second proteolytic cleavage. We also found that CA is an acylpeptide hydrolase rather than a previously expected acylamino acid acylase. It only exhibited exopeptidase activity for the hydrolysis of an externally added peptide, supporting the intra-molecular interaction. We propose that the final CA activation is an intramolecular process performed by an N-terminal nucleophile, during which large conformational changes in the ␣-subunit C-terminal region are required to bridge the gap between Glu 159 and Ser 170 .

Bifunctional quorum-quenching and antibiotic-acylase MacQ forms a 170-kDa capsule-shaped molecule containing spacer polypeptides

“…The self-cleaving maturation mechanism has been extensively studied using cephalosporin acylase (CA) 37 – 39 , 48 . Various SP-shortened mutants of CA were created 48 . They retained self-cleaving activity similar to wild-type CA.…”

Autoproteolytic Activation of ThnT Results in Structural Reorganization Necessary for Substrate Binding and Catalysis

“…Interestingly, prolonged incubation of ThnT resulted in further proteolysis of the α-subunit (Figure S1). This raised the possibility that a secondary cleavage, similar to that observed for CA, 26 might also occur for ThnT. The slow timescale of the apparent hydrolysis, however, suggested the phenomenon could be due to a contaminating protease.…”

A Straightforward Approach to Synthesize 7-Aminocephalosporanic Acid In Vivo in the Cephalosporin C Producer Acremonium chrysogenum

“…For enzymatic activation, autocatalytic cleavage of the CCA enzyme is necessary, where the precursor polypeptide is cut into an α-subunit and β-subunit through a series of hydrolytic reactions led by the Ser1β amino acid [ 19 , 55 , 56 , 57 ]. For CCAA and CCAB transformants, we succeeded in detecting proteins bands resembling the β-subunit (60 kDa), which emerged after cleavage.…”