Structural Basis for the Role of Asp-120 in Metallo-β-lactamases^,

Abstract

Metallo-beta-lactamases (mbetals) are zinc-dependent enzymes that hydrolyze a wide range of beta-lactam antibiotics. The mbetal active site features an invariant Asp-120 that ligates one of the two metal ions (Zn2) and a metal-bridging water/hydroxide (Wat1). Previous studies show that substitutions at Asp-120 dramatically affect mbetal activity, but no consensus exists as to its role in beta-lactam turnover. Here we present crystal structures of the Asn and Cys mutants of Asp-120 of the L1 mbetal from Stenotrophomonas maltophilia. Both mutants retain a dinuclear zinc center with Wat1 present. In the essentially inactive Cys enzyme Zn2 is displaced to a more buried position relative to that in the wild-type enzyme. In the catalytically impaired Asn enzyme the coordination of Zn2 is altered, neither it nor Wat1 is coordinated by Asn-120, and the N-terminal 19 amino acids, important to cooperative interactions between subunits in the wild-type enzyme, are disordered. Comparison with the structure of L1 complexed with the hydrolyzed oxacephem moxalactam suggests that in the Cys mutant Zn2 can no longer make stabilizing interactions with anionic nitrogen species formed in the hydrolytic reaction. The diminished activity of the Asn mutant arises from a combination of loss of intersubunit interactions and impaired proton transfer to, and reduced interaction of Zn2 with, the substrate amide nitrogen. We conclude that, while interactions of Asp-120 with active site water molecules are important to proton transfer and possibly nucleophilic attack by Wat1, its primary role is to optimally position Zn2 for catalytically important interactions with the charged amide nitrogen of substrate.

Structural Insights into the Subclass B3 Metallo-β-Lactamase SMB-1 and the Mode of Inhibition by the Common Metallo-β-Lactamase Inhibitor Mercaptoacetate

“…The value of the stereochemical parameter (37), which could discriminate between a square pyramid ( ϭ 0) and trigonal bipyramid ( ϭ 1), is 0.37 { ϭ (34). It must be pointed out that one of the carboxylate oxygen atoms of D120[76] is also involved in hydrogen bonding to the bridging hydroxide oxygen atom (Wat1), suggesting that D120[76] may be particularly important not only in orientating an OH Ϫ acting as a nucleophile to the carbonyl carbon on the ␤-lactam ring but also in positioning the Zn2 and H263 [215], as seen also in the subclass B1 and B3 MBLs (38)(39)(40). These results suggested that SMB-1 has the ability to keep the coordination geometry closer to the transition state through distortion of the regular geometry.…”

Crystal Structure of the Mobile Metallo-β-Lactamase AIM-1 from Pseudomonas aeruginosa: Insights into Antibiotic Binding and the Role of Gln157

“…Crystal structures for three other MBLs of subclass B3 are available (14,15,23,53). A comparison of the structure of AIM-1 with those previously determined structures is shown in Fig.…”

His224 Alters the R2 Drug Binding Site and Phe218 Influences the Catalytic Efficiency of the Metallo-β-Lactamase VIM-7

“…The resulting negatively charged tetrahedral intermediate is then stabilized by interactions with the metal, and Asp120 donates a proton to the nitrogen atom in the substrate, resulting in open-ing of the ␤-lactam ring (17). Several mutants of Asp120 in the class B3 MBL enzyme L1 from Stenotrophomonas maltophilia showed a 10-to 1,000-fold decrease in k cat compared to wild-type L1, and thus, Asp120 was confirmed to play a significant role in the hydrolysis reaction (22,23). This has also been shown for IMP-1 (24), CphA (25), and BcII (26).…”