Reference : Mutational analysis of VIM-2 reveals an essential determinant for metallo-beta-lactam...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/67948
Mutational analysis of VIM-2 reveals an essential determinant for metallo-beta-lactamase stability and folding.
English
Borgianni, Luisa [Università di Siena > Dipartimento di Biologia Molecolare > Laboratorio di Fisiologia e Biotecnologia dei Microrganismi > >]
Vandenameele, Julie mailto [Université de Liège - ULg > Département des sciences de la vie > Enzymologie et repliement des protéines >]
Matagne, André mailto [Université de Liège - ULg > Département des sciences de la vie > Enzymologie et repliement des protéines >]
Bini, Luca [Università di Siena > Dipartimento di Biologia Molecolare > Laboratorio di Fisiologia e Biotecnologia dei Microrganismi > >]
Bonomo, Robert A [Louis Stokes Cleveland > Department of Veterans Affairs Medical Center > > >]
Frère, Jean-Marie mailto [Université de Liège - ULg > > Centre d'ingénierie des protéines >]
Rossolini, Gian Maria mailto [Università di Siena > Dipartimento di Biologia Molecolare > Laboratorio di Fisiologia e Biotecnologia dei Microrganismi > >]
Docquier, Jean*-Denis [Università di Siena > Dipartimento di Biologia Molecolare > Laboratorio di Fisiologia e Biotecnologia dei Microrganismi > >]
2010
Antimicrobial Agents and Chemotherapy
American Society for Microbiology (ASM)
54
8
3197-204
Yes (verified by ORBi)
International
0066-4804
1098-6596
Washington
DC
[en] metallo-beta-lactamase ; saturation mutagenesis ; folding ; catalytic activity
[en] Metallo-beta-lactamase (MBL)-producing bacteria are emerging worldwide and represent a formidable threat to the efficacy of relevant beta-lactams, including carbapenems, expanded-spectrum cephalosporins, and beta-lactamase inactivator/beta-lactam combinations. VIM-2 is currently the most widespread MBL and represents a primary target for MBL inhibitor research, the clinical need for which is expected to further increase in the future. Using a saturation mutagenesis approach, we probed the importance of four residues (Phe-61, Ala-64, Tyr-67, and Trp-87) located close to the VIM-2 active site and putatively relevant to the enzyme activity based on structural knowledge of the enzyme and on structure-activity relationships of the subclass B1 MBLs. The ampicillin MIC values shown by the various mutants were affected very differently depending on the randomized amino acid position. Position 64 appeared to be rather tolerant to substitution, and kinetic studies showed that the A64W mutation did not significantly affect substrate hydrolysis or binding, representing an important difference from IMP-type enzymes. Phe-61 and Tyr-67 could be replaced with several amino acids without the ampicillin MIC being significantly affected, but in contrast, Trp-87 was found to be critical for ampicillin resistance. Further kinetic and biochemical analyses of W87A and W87F variants showed that this residue is apparently important for the structure and proper folding of the enzyme but, surprisingly, not for its catalytic activity. These data support the critical role of residue 87 in the stability and folding of VIM-2 and might have strong implications for MBL inhibitor design, as this residue would represent an ideal target for interaction with small molecules.
http://hdl.handle.net/2268/67948
10.1128/AAC.01336-09

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