Catalytic Mechanism and Evolution of Metallo-beta-Lactamases(conferencista invitado)

ALEJANDRO J. VILA, LETICIA I. LLARRULL, MARIANA F. TIONI, PABLO E. TOMATIS, VALERIA CAMPOS BERMUDEZ, JAVIER M. GONZÁLEZ, LISANDRO J. GONZÁLEZ, MA. EUGENIA ZABALLA, MA. NATALIA LISA AND LUCIANO A. ABRIATA

Tucumán, Argentina

Congreso; XXVII Congreso Argentino de Química “Dr. Pedro J. Aymonino"; 2008

Asociación Química Argentina

<!-- /* Font Definitions */ @font-face {font-family:Times; panose-1:2 2 6 3 5 4 5 2 3 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:536902279 -2147483648 8 0 511 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; mso-bidi-font-size:10.0pt; font-family:Times; mso-fareast-font-family:"Times New Roman"; mso-bidi-font-family:"Times New Roman"; mso-ansi-language:EN-US; mso-fareast-language:EN-US;} p.AbstractBody, li.AbstractBody, div.AbstractBody {mso-style-name:"Abstract Body"; mso-style-update:auto; margin:0cm; margin-bottom:.0001pt; text-align:justify; line-height:150%; mso-pagination:widow-orphan; tab-stops:22.0pt; font-size:12.0pt; mso-bidi-font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-US; mso-fareast-language:EN-US;} @page Section1 {size:595.0pt 842.0pt; margin:3.0cm 3.0cm 3.0cm 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Beta-lactamases represent the prevalent resistance mechanism of bacteria toward beta-lactam antibiotics. Metallo-beta-lactamases (MBLs) are zinc-dependent enzymes able to hydrolyze all beta-lactam based drugs. Sequence analysis of MBLs reveals a large structural diversity, that gives rise to the presence of mono- and dinuclear metal sites. MBLs have been recently subdivided into classes B1, B2 and B3, each of them displaying different zinc ligands and coordination geometries. We have studied the B1 MBL from B.cereus (BcII) and analyzed the minimal structural requirements for activity in these enzymes. This enzyme has been proposed to be active both in its mono- and dinuclear forms. The selective elimination of each metal site by mutagenesis of the ligands gives rise mononuclear derivatives showing very poor catalytic efficiencies. Rapid kinetics studies in the Co(II)-substituted enzyme have allowed us to trap a reaction intermediate with a unique resonant structure. Directed evolution on BcII by DNA shuffling resulted in a expanded substrate spectrum of this enzyme, without sacrificing its stability nor the hydrolytic efficiency towards classical substrates of BcII. The mutations that give rise to these effects parallel others naturally found in MBL´s from pathogenic bacteria, and are related to the second-shell ligands of the second zinc ion, expected to play a supramolecular control of reactivity.