title: 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 αβ 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 first-order, intra-molecular process. Glutaryl-7-aminocephalosporanic acid is the classic substrate of CA, in which the N-terminal Ser(170) of the β-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 intra-molecular process performed by an N-terminal nucleophile, during which large conformational changes in the α-subunit C-terminal region are required to bridge the gap between Glu(159) and Ser(170).
authors.affiliation: State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Yin
authors.forename: Zixin
authors.initials: Z
authors.lastname: Deng
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhao
authors.forename: Xi
authors.initials: X
authors.lastname: Huang
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002511:Cephalosporins / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D020125:Mutation, Missense; D010399:Penicillin Amidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D012694:Serine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002511:Cephalosporins; D004792:Enzyme Precursors; C081063:glutaryl-7-aminocephalosporanic acid; D012694:Serine; D010399:Penicillin Amidase