This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, Y. S.
Right arrow Articles by Park, S. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, Y. S.
Right arrow Articles by Park, S. S.

 Previous Article  |  Next Article 

Journal of Bacteriology, September 1998, p. 4576-4582, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Two-Step Autocatalytic Processing of the Glutaryl 7-Aminocephalosporanic Acid Acylase from Pseudomonas sp. Strain GK16

Young Sik Lee and Sung Soo Park*

Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea

Received 19 March 1998/Accepted 22 June 1998

The glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase of Pseudomonas sp. strain GK16 is an (alpha beta )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).

* Corresponding author. Mailing address: Graduate School of Biotechnology, Korea University, Seoul, Korea 136-701. Phone: 8202-3290-3431. Fax: 8202-929-1864. E-mail: sspark{at}kuccnx.korea.ac.kr.

Journal of Bacteriology, September 1998, p. 4576-4582, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Okada, T., Suzuki, H., Wada, K., Kumagai, H., Fukuyama, K. (2007). Crystal Structure of the {gamma}-Glutamyltranspeptidase Precursor Protein from Escherichia coli: STRUCTURAL CHANGES UPON AUTOCATALYTIC PROCESSING AND IMPLICATIONS FOR THE MATURATION MECHANISM. J. Biol. Chem. 282: 2433-2439 [Abstract] [Full Text]  
  • Sio, C. F., Otten, L. G., Cool, R. H., Diggle, S. P., Braun, P. G., Bos, R., Daykin, M., Camara, M., Williams, P., Quax, W. J. (2006). Quorum Quenching by an N-Acyl-Homoserine Lactone Acylase from Pseudomonas aeruginosa PAO1. Infect. Immun. 74: 1673-1682 [Abstract] [Full Text]  
  • Kim, J. K., Yang, I. S., Shin, H. J., Cho, K. J., Ryu, E. K., Kim, S. H., Park, S. S., Kim, K. H. (2006). Insight into autoproteolytic activation from the structure of cephalosporin acylase: A protein with two proteolytic chemistries. Proc. Natl. Acad. Sci. USA 103: 1732-1737 [Abstract] [Full Text]  
  • Yoon, J., Oh, B., Kim, K., Park, J., Han, D., Kim, K. K., Cha, S.-S., Lee, D., Kim, Y. (2004). A Bound Water Molecule Is Crucial in Initiating Autocatalytic Precursor Activation in an N-terminal Hydrolase. J. Biol. Chem. 279: 341-347 [Abstract] [Full Text]  
  • Suzuki, H., Kumagai, H. (2002). Autocatalytic Processing of gamma -Glutamyltranspeptidase. J. Biol. Chem. 277: 43536-43543 [Abstract] [Full Text]  
  • Kim, S., Kim, Y. (2001). Active Site Residues of Cephalosporin Acylase Are Critical Not Only for Enzymatic Catalysis but Also for Post-translational Modification. J. Biol. Chem. 276: 48376-48381 [Abstract] [Full Text]  
  • Lee, Y. S., Kim, H. W., Park, S. S. (2000). The Role of alpha -Amino Group of the N-terminal Serine of beta Subunit for Enzyme Catalysis and Autoproteolytic Activation of Glutaryl 7-Aminocephalosporanic Acid Acylase. J. Biol. Chem. 275: 39200-39206 [Abstract] [Full Text]  
  • Kim, Y., Kim, S., Earnest, T. N., Hol, W. G. J. (2002). Precursor Structure of Cephalosporin Acylase. INSIGHTS INTO AUTOPROTEOLYTIC ACTIVATION IN A NEW N-TERMINAL HYDROLASE FAMILY. J. Biol. Chem. 277: 2823-2829 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»