THE EFFECT ON PEPTIDOGLYCAN COMPOSITION OF UNCHARACTERIZED PAE-AMPC MUTANTS PROBES ITS FUNCTIONALITY AS DD-PEPTIDASE

A. ROPY¹, J.A. AYALA²*
¹a.Centro de Biología Molecular Severo Ochoa, Madrid, Spain, b.Chemistry Department, Faculty of Science, Fayoum University, 63514, Egypt [Current Address]
²Centro de Biología Molecular Severo Ochoa, Madrid, Spain
* Corresponding Author : jayala@cbm.csic.es

Received : 29-10-2015     Accepted : 19-11-2015     Published : 07-12-2015
Volume : 7     Issue : 6       Pages : 710 - 716
Int J Microbiol Res 7.6 (2015):710-716

Keywords : AmpC beta-lactamase, Endopeptidase, Carboxypepetidase, DD-peptidases, Low-molecular-mass penicillin-binding proteins, Peptidoglycan, Pseudomonas aeruginosa
Academic Editor : Patricia A. Cordobab, Juan Manuel Tovar-pedraza, Dong Wook Kim, Juan A. Ayala, Guillermo H. Sguazza, Liliana S. Losch, Natalia Y. Picco, Maria L. Castrillo
Conflict of Interest : None declared
Acknowledgements/Funding : This work was supported by grantsBFU2009-09200 from the Ministerio de Economía y Competitividad of Spain and 223431 DIVINOCELL from the European Union. Predoctoral grant JAE/Predoc from the Consejo Superior de Investigaciones Cientificas to A.R. is acknowledged.
Author Contribution : None declared

It has been largely hypothesized, but never probed, that PBPs and β-lactamases come from an ancient common ancestor, although, there are examples of cross enzymatic reactions (DD-peptidase and β-lactamase) for both types of enzymes. This work aimed to characterize the effect of point mutations [R2→G (AmpC-F2), P243→L (AmpC-F4:C3) and I51→T (AmpC-F4:C6)] on β-lactamase activity of AmpC (Pae-AmpC) from Pseudomonas aeruginosa PAO1 strain; also to track the effect of AmpC activity on peptidoglycan composition, as a consequence of DD-peptidase activities . So, periplasmic and cytoplasmic forms of these Pae-AmpC mutants and the wild type Pae-AmpC were cloned, purified by Ni-affinity chromatography, and then tested for their β-lactamase activities and their effect on PG composition from wild type and mutants of E. coli and P. aeruginosa. In vitro assay for β-lactamase activities showed that both point mutations P243→L and I51→T caused 5-fold decrease, while R2→G change caused 2.5-fold decrease in β-lactamase activity when compared with AmpC-F4. On the other hand the cytoplasmic form (AmpC-F3) displayed 8-fold increase in β-lactamase activity. Moreover, AmpC-F3 displayed a secondary DD-endopeptidase/DD-carboxypeptidase on the whole PG in vitro, and DD-endopeptidase activity on individual purified muropeptides. Data obtained from HPLC analysis of PG composition support previous suggestions that AmpC can elicit DD-carboxypeptidase or DD-endopeptidase activity most probably due to structural similarities of the active site with DD-peptidase enzymes having these activities (LMM-PBPs).