title: Modeling domino effects in enzymes: molecular basis of the substrate specificity of the bacterial metallo-beta-lactamases IMP-1 and IMP-6.
abstract: Metallo-beta-lactamases can hydrolyze a broad spectrum of beta-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.
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Oelschlaeger
authors.forename: Rolf D
authors.initials: RD
authors.lastname: Schmid
authors.forename: Juergen
authors.initials: J
authors.lastname: Pleiss
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D019295:Computational Biology / Q000379:methods; D003198:Computer Simulation; D004795:Enzyme Stability / Q000235:genetics; D005998:Glycine / Q000235:genetics; D006358:Hot Temperature; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics
chemical_list: D012694:Serine; C409201:beta-lactamase IMP-1; D001618:beta-Lactamases; D005998:Glycine