Abstract
Cephalosporin acylase is a member of the N-terminal hydrolase family, which is activated from an inactive precursor by autoproteolytic processing to generate a new N-terminal nucleophile Ser or Thr. The gene structure of the precursor cephalosporin acylases generally consists of a signal peptide that is followed by an alpha-subunit, a spacer sequence, and a beta-subunit. The cephalosporin acylase precursor is post-translationally modified into an active heterodimeric enzyme with alpha- and beta-subunits, first by intramolecular cleavage and, second, by intermolecular cleavage. Intramolecular autocatalytic proteolysis is initiated by nucleophilic attack of the residue Ser-1beta onto the adjacent scissile carbonyl carbon. This study determined the precursor structure after disabling the intramolecular cleavage. This study also provides experimental evidence showing that a conserved water molecule plays an important role in assisting the polarization of the OG atom of Ser-1beta to generate a strong nucleophile and to direct the OG atom of the Ser-1beta to a target carbonyl carbon. Intramolecular proteolysis is disabled as a result of a mutation of the residues causing conformational distortion to the active site. This is because distortion affects the existence of the catalytically crucial water at the proper position. This study provides the first evidence showing that a bound water molecule plays a critical role in initiating intramolecular cleavage in the post-translational modification of the precursor enzyme.
Highlights
Cephalosporin acylase (CA)1 is a new family of the N-terminal (Ntn) hydrolase superfamily that is defined by the SCOP data base (1)
Site-directed Mutagenesis of CA from Pseudomonas diminuta KAC-1 (CAD) Yields a Different Status of Intramolecular and Intermolecular Cleavage—The CAD precursor of the wild-type spontaneously conducts intramolecular cleavage by autocatalytic proteolysis at the scissile peptide bond between the Gly-169s and Ser-1 residues upon folding after protein synthesis
In particular the mutation of Ser-1 to Ala (S1A) completely lost its intramolecular autoproteolytic activity (24) because the OG atom from the side chain of Ser-1 plays an important role in nucleophilic attack at the scissile peptide bond
Summary
The structures of CA from Pseudomonas diminuta KAC-1 (CAD) and a precursor CAD were determined (12, 13). A previous structural study of the precursor, CAD, revealed that conserved water (present in both the precursor CAD and the mature CAD) plays an important role in assisting the OG atom of Ser-1 so that the nucleophilic OG atom can carry out an attack on the scissile carbonyl carbon of Gly-169s (13). The precursor structure that was disabled in the autocatalytic proteolysis lost the bound water from the active site. A comparison of the active sites between the two different precursors with and without the autocatalytic proteolysis provided data that showed that the absence of bound water abolished the capability of carrying out the autoproteolytic intramolecular cleavage from the precursor CA. This study provides the first evidence that a bound water molecule can play a critical role in initiating the beginning of intramolecular cleavage in the post-translational modification of the precursor enzyme
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