Two-step autocatalytic processing of the glutaryl 7-aminocephalosporanic acid acylase from Pseudomonas sp. strain GK16.
The glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase of Pseudomonas sp. strain GK16 is an (alphabeta)2 heterotetramer of two nonidentical subunits. These subunits are derived from nascent polypeptides that are cleaved proteolytically between Gly198 and Ser199 after the nascent polypeptides have been translocated into the periplasm. The activation mechanism of the GL-7-ACA acylase has been analyzed by both in vivo and in vitro expression studies, site-directed mutagenesis, in vitro renaturation of inactive enzyme precursors, and enzyme reconstitution. An active enzyme complex was found in the cytoplasm when its translocation into the periplasm was suppressed. In addition, the in vitro-expressed GL-7-ACA acylase was processed into alpha and beta subunits, and the inactive enzyme aggregate of the precursor was also processed and became active during the renaturation step. Mutation of Ser199 to Cys199 and enzyme reconstitution allowed us to identify the secondary processing site that resides in the alpha subunit and to show that Ser199 of the beta subunit is essential for these two sequential processing steps. Mass spectrometry clearly indicated that the secondary processing occurs at Gly189-Asp190. All of the data suggest that the enzyme is activated through a two-step autocatalytic process upon folding: the first step is an intramolecular cleavage of the precursor between Gly198 and Ser199 for generation of the alpha subunit, containing the spacer peptide, and the beta subunit; the second is an intermolecular event, which is catalyzed by the N-terminal Ser (Ser199) of the beta subunit and results in a further cleavage and the removal of the spacer peptide (Asp190 to Gly198).
Lee
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YS
Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea.
Park
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SS
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002384:Catalysis; D004269:DNA, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010399:Penicillin Amidase; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D012694:Serine; D000581:Amidohydrolases; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase