Crystal Structures of Pseudomonas aeruginosa GIM-1: Active-Site Plasticity in Metallo-β-Lactamases

Abstract

Metallo-␤-lactamases (MBLs) have rapidly disseminated worldwide among clinically important Gram-negative bacteria and have challenged the therapeutic use of ␤-lactam antibiotics, particularly carbapenems. The bla GIM-1 gene, encoding one such enzyme, was first discovered in a Pseudomonas aeruginosa isolate from 2002 and has more recently been reported in Enterobacteriaceae. Here, we present crystal structures of GIM-1 in the apo-zinc (metal-free), mono-zinc (where Cys221 was found to be oxidized), and di-zinc forms, providing nine independently refined views of the enzyme. GIM-1 is distinguished from related MBLs in possessing a narrower active-site groove defined by aromatic side chains (Trp228 and Tyr233) at positions normally occupied by hydrophilic residues in other MBLs. Our structures reveal considerable flexibility in two loops (loop 1, residues 60 to 66; loop 2, residues 223 to 242) adjacent to the active site, with open and closed conformations defined by alternative hydrogen-bonding patterns involving Trp228. We suggest that this capacity for rearrangement permits GIM-1 to hydrolyze a wide range of ␤-lactams in spite of possessing a more constrained active site. Our results highlight the structural diversity within the MBL enzyme family.

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

“…We therefore conclude that residue 224 in the L3 loop is an important residue determinant for both stability and enzyme activity for the VIM family of metallo-␤-lactamases. During catalysis, the L3 loop of VIM-7 is most likely flexible in a fashion similar to that found for the L3 loop of GIM-1 (27). Still, the interplay between residues 224 and 228 in the L3 loop and 67 in the L1 loop also seems important for activity.…”

“…Based on the VIM-7 crystal structure, we hypothesized that His224 and Phe218 were involved in the observed reduced catalytic efficiency (16). Furthermore, the L3 loop, consisting of residues 223 to 240, has been shown to play a role in B1 MBLs (27,31,44). Residue 224 was found to be a lysine in many B1 MBLs (33), and the positively charged side chain is thought to be important for both binding and positioning of substrate (45) to various degrees (33,46,47).…”

“…Two anti-parallel ␤-strands connected by a loop determine the L1 loop (residues 60 to 66) and are proposed to form a lid over the active site in several class B1 MBLs, including GIM-1 (27), VIM-7 (16), and IMP-1 (28)(29)(30). For the latter, the mutation of F218Y increased the catalytic efficiency compared to that of wild-type IMP-1 through hydrogen bond formation, improving the closingdown movement of this lid (28,29).…”

Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding

“…The two L1 loops of chain A and chain B are facing each other within the asymmetric unit and are thus involved in the TMB-1 crystal packing. Residues in the L3 loop are involved in substrate binding, and both the L1 and L3 loops have been shown to be flexible in, e.g., GIM-1 (18).…”

“…In TMB-1, serine (S) and glutamic acid (E) are located at positions 117 and 119, respectively, similarly to other MBLs, e.g., GIM-1 (18). IMP-1 and NDM-1 have serine and glutamine (Q), respectively, at position 119 (14).…”