THE CRYSTAL-STRUCTURE OF THE L1 METALLO-BETA-LACTAMASE FROM STENOTROPHOMONAS-MALTOPHILIA AT 1.7 ANGSTROM RESOLUTION

The structure of the L1 metallo-beta-lactamase from the opportunistic pathogen Stenotrophomonas maltophilia has been determined at 1.7 Angst rom resolution by the multiwavelength anomalous dispersion (MAD) appro ach exploiting both the intrinsic binuclear zinc centre and incorporat ed selenomethionine residues. L1 is unique amongst all known beta-lact amases in that it exists as a tetramer. The protein exhibits the alpha beta/beta alpha fold found only in the metallo-beta-lactamases and di splays several unique features not previously observed in these enzyme s. These include a disulphide bridge and two substantially elongated l oops connected to the active site of the enzyme. Two closely spaced zi nc ions are bound at the active site with tetrahedral (Zn1) and trigon al bipyramidal (Zn2) co-ordination, respectively; these are bridged by a water molecule which we propose acts as the nucleophile in the hydr olytic reaction. Ligation of the second zinc ion involves both residue s and geometry which have not been previously observed in the metallo- beta-lactamases. Simulated binding of the substrates ampicillin, cefta zidime 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 nitroge n with the-zinc ions and of the beta-lactam carboxylate with Ser187. W e describe a catalytic mechanism whose principal features are a nucleo philic attack of the bridging water on the beta-lactam carbonyl carbon , electrostatic stabilisation of a negatively charged tetrahedral tran sition state and protonation of the beta-lactam nitrogen by a second w ater molecule co-ordinated by Zn2. Further, we propose that direct met al:substrate interactions provide a substantial contribution to substr ate binding and that this may explain the lack of specificity which is a feature of this class of enzyme. (C) 1998 Academic Press.