Metallo-
-lactamases are zinc-dependent enzymes responsible for resistance to
-lactamantibiotics in a variety of host bacteria, usually Gram-negative species that act as opportunist pathogens.They hydrolyze all classes of
-lactam antibiotics, including carbapenems, and escape the action of available
-lactamase inhibitors. Efforts to develop effective inhibitors have been hampered by the lack of structuralinformation regarding how these enzymes recognize and turn over
-lactam substrates. We report herethe crystal structure of the
Stenotrophomonas maltophilia L1 enzyme in complex with the hydrolysis productof the 7
-methoxyoxacephem, moxalactam. The on-enzyme complex is a 3'-
exo-methylene speciesgenerated by elimination of the 1-methyltetrazolyl-5-thiolate anion from the 3'-methyl group. Moxalactambinding to L1 involves direct interaction of the two active site zinc ions with the
-lactam amide and C4carboxylate, groups that are common to all
-lactam substrates. The 7
-[(4-hydroxyphenyl)malonyl]-aminosubstituent makes limited hydrophobic and hydrogen bonding contacts with the active site groove. Themode of binding provides strong evidence that a water molecule situated between the two metal ions isthe most likely nucleophile in the hydrolytic reaction. These data suggest a reaction mechanism for metallo-
-lactamases in which both metal ions contribute to catalysis by activating the bridging water/hydroxidenucleophile, polarizing the substrate amide bond for attack and stabilizing anionic nitrogen intermediates.The structure illustrates how a binuclear zinc site confers upon metallo-
-lactamases the ability both torecognize and efficiently hydrolyze a wide variety of
-lactam substrates.
