3s8r Citations

Crystal structures of glutaryl 7-aminocephalosporanic acid acylase: insight into autoproteolytic activation.

Biochemistry 42 4084-93 (2003)
Cited: 27 times
EuropePMC logo PMID: 12680762


Glutaryl 7-aminocephalosporanic acid acylase (GCA, EC is a member of N-terminal nucleophile (Ntn) hydrolases. The native enzyme is an (alpha beta)(2) heterotetramer originated from an enzymatically inactive precursor of a single polypeptide. The activation of precursor GCA consists of primary and secondary autoproteolytic cleavages, generating a terminal residue with both a nucleophile and a base and releasing a nine amino acid spacer peptide. We have determined the crystal structures of the recombinant selenomethionyl native and S170A mutant precursor from Pseudomonas sp. strain GK16. Precursor activation is likely triggered by conformational constraints within the spacer peptide, probably inducing a peptide flip. Autoproteolytic site solvent molecules, which have been trapped in a hydrophobic environment by the spacer peptide, may play a role as a general base for nucleophilic attack. The activation results in building up a catalytic triad composed of Ser170/His192/Glu624. However, the triad is not linked to the usual hydroxyl but the free alpha-amino group of the N-terminal serine residue of the native GCA. Mutagenesis and structural data support the notion that the stabilization of a transient hydroxazolidine ring during autoproteolysis would be critical during the N --> O acyl shift. The autoproteolytic activation mechanism for GCA is described.

Reviews citing this publication (2)

  1. Cephalosporin C acylase: dream and(/or) reality. Pollegioni L, Rosini E, Molla G. Appl. Microbiol. Biotechnol. 97 2341-2355 (2013)
  2. Bacterial β-aminopeptidases: structural insights and applications for biocatalysis. Heck T, Geueke B, Kohler HP. Chem. Biodivers. 9 2388-2409 (2012)

Articles citing this publication (25)

  1. Quorum quenching by an N-acyl-homoserine lactone acylase from Pseudomonas aeruginosa PAO1. Sio CF, Otten LG, Cool RH, Diggle SP, Braun PG, Bos R, Daykin M, Cámara M, Williams P, Quax WJ. Infect. Immun. 74 1673-1682 (2006)
  2. The MEROPS batch BLAST: a tool to detect peptidases and their non-peptidase homologues in a genome. Rawlings ND, Morton FR. Biochimie 90 243-259 (2008)
  3. Overexpression, one-step purification, and biochemical characterization of a recombinant gamma-glutamyltranspeptidase from Bacillus licheniformis. Lin LL, Chou PR, Hua YW, Hsu WH. Appl. Microbiol. Biotechnol. 73 103-112 (2006)
  4. Structural constraints on protein self-processing in L-aspartate-alpha-decarboxylase. Schmitzberger F, Kilkenny ML, Lobley CM, Webb ME, Vinkovic M, Matak-Vinkovic D, Witty M, Chirgadze DY, Smith AG, Abell C, Blundell TL. EMBO J. 22 6193-6204 (2003)
  5. Insight into autoproteolytic activation from the structure of cephalosporin acylase: a protein with two proteolytic chemistries. Kim JK, Yang IS, Shin HJ, Cho KJ, Ryu EK, Kim SH, Park SS, Kim KH. Proc. Natl. Acad. Sci. U.S.A. 103 1732-1737 (2006)
  6. Crystallographic snapshot of glycosylasparaginase precursor poised for autoprocessing. Wang Y, Guo HC. J. Mol. Biol. 403 120-130 (2010)
  7. Insights into cis-autoproteolysis reveal a reactive state formed through conformational rearrangement. Buller AR, Freeman MF, Wright NT, Schildbach JF, Townsend CA. Proc. Natl. Acad. Sci. U.S.A. 109 2308-2313 (2012)
  8. SEA domain autoproteolysis accelerated by conformational strain: energetic aspects. Sandberg A, Johansson DG, Macao B, Härd T. J. Mol. Biol. 377 1117-1129 (2008)
  9. The three-dimensional structure of the extracellular adhesion domain of the sialic acid-binding adhesin SabA from Helicobacter pylori. Pang SS, Nguyen ST, Perry AJ, Day CJ, Panjikar S, Tiralongo J, Whisstock JC, Kwok T. J. Biol. Chem. 289 6332-6340 (2014)
  10. Autoproteolytic activation of human aspartylglucosaminidase. Saarela J, Oinonen C, Jalanko A, Rouvinen J, Peltonen L. Biochem. J. 378 363-371 (2004)
  11. Structure of a class III engineered cephalosporin acylase: comparisons with class I acylase and implications for differences in substrate specificity and catalytic activity. Golden E, Paterson R, Tie WJ, Anandan A, Flematti G, Molla G, Rosini E, Pollegioni L, Vrielink A. Biochem. J. 451 217-226 (2013)
  12. Crystallographic snapshot of a productive glycosylasparaginase-substrate complex. Wang Y, Guo HC. J. Mol. Biol. 366 82-92 (2007)
  13. Mutational analysis of a key residue in the substrate specificity of a cephalosporin acylase. Otten LG, Sio CF, van der Sloot AM, Cool RH, Quax WJ. Chembiochem 5 820-825 (2004)
  14. A highly active adipyl-cephalosporin acylase obtained via rational randomization. Otten LG, Sio CF, Reis CR, Koch G, Cool RH, Quax WJ. FEBS J. 274 5600-5610 (2007)
  15. Autoproteolytic and catalytic mechanisms for the β-aminopeptidase BapA--a member of the Ntn hydrolase family. Merz T, Heck T, Geueke B, Mittl PR, Briand C, Seebach D, Kohler HP, Grütter MG. Structure 20 1850-1860 (2012)
  16. The N-terminal nucleophile serine of cephalosporin acylase executes the second autoproteolytic cleavage and acylpeptide hydrolysis. Yin J, Deng Z, Zhao G, Huang X. J. Biol. Chem. 286 24476-24486 (2011)
  17. Residues Arg114 and Arg337 are critical for the proper function of Escherichia coli gamma-glutamyltranspeptidase. Ong PL, Yao YF, Weng YM, Hsu WH, Lin LL. Biochem. Biophys. Res. Commun. 366 294-300 (2008)
  18. Initial insight into the function of the lysosomal 66.3 kDa protein from mouse by means of X-ray crystallography. Lakomek K, Dickmanns A, Kettwig M, Urlaub H, Ficner R, Lübke T. BMC Struct. Biol. 9 56 (2009)
  19. Effects of C-terminal truncation on autocatalytic processing of Bacillus licheniformis gamma-glutamyl transpeptidase. Chang HP, Liang WC, Lyu RC, Chi MC, Wang TF, Su KL, Hung HC, Lin LL. Biochemistry Mosc. 75 919-929 (2010)
  20. Structural features of cephalosporin acylase reveal the basis of autocatalytic activation. Cho KJ, Kim JK, Lee JH, Shin HJ, Park SS, Kim KH. Biochem. Biophys. Res. Commun. 390 342-348 (2009)
  21. Production of autoproteolytically subunit-assembled 7-beta-(4-carboxybutanamido)cephalosporanic acid (GL-7ACA) acylase from Pseudomonas sp. C427 using a chitin-binding domain. Nagao K, Yamashita M, Ueda M. Appl. Microbiol. Biotechnol. 65 407-413 (2004)
  22. Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants. Chandra PM, Brannigan JA, Prabhune A, Pundle A, Turkenburg JP, Dodson GG, Suresh CG. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 61 124-127 (2005)
  23. Experimental evidence for the involvement of amino acid residue Glu398 in the autocatalytic processing of Bacillus licheniformis γ-glutamyltranspeptidase. Chi MC, Chen YY, Lo HF, Lin LL. FEBS Open Bio 2 298-304 (2012)
  24. Alteration of substrate selection of antibiotic acylase from β-lactam to echinocandin. Isogai Y, Nakayama K. Protein Eng. Des. Sel. 29 49-56 (2016)
  25. Bifunctional quorum-quenching and antibiotic-acylase MacQ forms a 170-kDa capsule-shaped molecule containing spacer polypeptides. Yasutake Y, Kusada H, Ebuchi T, Hanada S, Kamagata Y, Tamura T, Kimura N. Sci Rep 7 8946 (2017)