The crystal structure of the L1 metallo-β-lactamase from Stenotrophomonas maltophilia at 1.7 Å resolution

The structure of the L1 metallo-beta-lactamase from the opportunistic pathogen Stenotrophomonas maltophilia has been determined at 1.7 Angstrom resolution by the multiwavelength anomalous dispersion (MAD) approach exploiting both the intrinsic binuclear zinc centre and incorporated selenomethionine residues. L1 is unique amongst all known beta-lactamases in that it exists as a tetramer. The protein exhibits the alpha beta/beta alpha fold found only in the metallo-beta-lactamases and displays several unique features not previously observed in these enzymes. These include a disulphide bridge and two substantially elongated loops connected to the active site of the enzyme. Two closely spaced zinc ions are bound at the active site with tetrahedral (Zn1) and trigonal bipyramidal (Zn2) co-ordination, respectively; these are bridged by a water molecule which we propose acts as the nucleophile in the hydrolytic reaction. Ligation of the second zinc ion involves both residues and geometry which have not been previously observed in the metallo-beta-lactamases. Simulated binding of the substrates ampicillin, ceftazidime and imipenem suggests that the substrate is able to bind to the enzyme in a variety of different conformations whose common features are direct interactions of the beta-lactam carbonyl oxygen and nitrogen with the-zinc ions and of the beta-lactam carboxylate with Ser187. We describe a catalytic mechanism whose principal features are a nucleophilic attack of the bridging water on the beta-lactam carbonyl carbon, electrostatic stabilisation of a negatively charged tetrahedral transition state and protonation of the beta-lactam nitrogen by a second water molecule co-ordinated by Zn2. Further, we propose that direct metal:substrate interactions provide a substantial contribution to substrate binding and that this may explain the lack of specificity which is a feature of this class of enzyme. (C) 1998 Academic Press.