Modeling Domino Effects in Enzymes: Molecular Basis of the Substrate Specificity of the Bacterial Metallo-β-lactamases IMP-1 and IMP-6

Abstract

Metallo-β-lactamases can hydrolyze a broad spectrum of β-lactam antibiotics and thus confer resistance to bacteria. For the Pseudomonas aeruginosa enzyme IMP-1, several variants have been reported. IMP-6 and IMP-1 differ by a single residue (glycine and serine at position 196, respectively), but have significantly different substrate spectra; while the catalytic efficiency toward the two cephalosporins cephalothin and cefotaxime is similar for both variants, IMP-1 is up to 10-fold more efficient than IMP-6 toward cephaloridine and ceftazidime. Interestingly, this biochemical effect is caused by a residue remote from the active site. The substrate-specific impact of residue 196 was studied by molecular dynamics simulations using a cationic dummy atom approach for the zinc ions. Substrates were docked in an intermediate structure near the transition state to the binding site of IMP-1 and IMP-6. At a simulation temperature of 100 K, most complexes were stable during 1 ns of simulation time. However, at higher temperatures, some complexes became unstable and the substrate changed to a nonactive conformation. To model stability, six molecular dynamics simulations at 100 K were carried out for all enzyme−substrate complexes. Stable structures were further heated to 200 and 300 K. By counting stable structures, we derived a stability ranking score which correlated with experimentally determined catalytic efficiency. The use of a stability score as an indicator of catalytic efficiency of metalloenzymes is novel, and the study of substrates in a near-transition state intermediate structure is superior to the modeling of Michaelis complexes. The remote effect of residue 196 can be described by a domino effect:  upon replacement of serine with glycine, a hole is created and a stabilizing interaction between Ser196 and Lys33 disappears, rendering the neighboring residues more flexible; this increased flexibility is then transferred to the active site.

Additional Information

© 2003 American Chemical Society. Received 4 February 2003. Published online 8 July 2003. Published in print 1 August 2003. This work was supported by the German Federal Ministry of a Education and Research (Project PTJ 31/0312702). We thank Florian Barth, Institute of Technical Biochemistry, University of Stuttgart, for LINUX cluster maintenance and assistance with Amber 6.0, and acknowledge the valuable suggestions by the referee regarding the Pro32−His197 interaction.

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