1. Binding and Catalysis of Metallo-  -Lactamases Studied using a SCC-DFTB/Charmm Approach D. Xu and H. Guo Department of Chemistry University of New Mexico

2. Why computational biochemistry? Experimental data alone do not provide complete picture. Understand interactions, mechanisms, and dynamics of substrate binding and enzyme catalysis at molecular level. Help design new and more efficient inhibitors and catalysts.

3. Metallo-  -lactamases One of four classes (B) of bacterial hydrolases responsible for penicillin resistance. Broad substrate spectrum. No clinically useful inhibitors. Rapid spreading between species via plasmid and integron- borne mechanisms. CphA L1

4. Challenges of metallo-enzymes Very difficult to model using force fields, because metal-ligand bonds are neither pure electrostatic nor covalent. Quantum chemical treatments include a necessarily large number of atoms Reaction mechanisms are often complex.

5. Computational Model To retain the correct electrostatic and van der Waals micro-environment, it has to include protein residues and solvent waters. To be able to describe bond forming and breaking processes, it has to use quantum mechanical potential.

6. Compromise: QM/MM method QM potential for reaction region. MM force field for surrounding and solvent. Boundary. MM QM Enzyme Substrate

7. QM/MM Hamiltonian Effective Valence Bond Semi-empirical MO Semi-empirical DFT Ab initio MO DFT CHARMM Amber Charge-charge Lennard-Jones Link atoms GHO Pseudo-bond

8. QM/MM Self-consistent charge density functional tight binding (SCC-DFTB) for QM region (substrate, metal cofactors and their ligands). CHARMM all atom force field for MM region. TIP3P model for solvent water. Link atoms at the boundary.

9. SCC-DFTB Approximate DFT method. Highly efficient, allow statistical sampling. More accurate than AM1 and PM3, particularly for zinc enzymes. Better description of H-bonds. Parameters exist for HCONS and biological Zn(II) ion. Validated in many biological systems. Q. Cui, 2006

10. CphA from A. hydrophila B2 subclass. Highly specific to carbapenems. Only active with single Zn co- factor, while second Zn ion inhibits enzyme. Structures of apo enzyme and complex with intermediate determined in 2005.

11. Hydrolysis of biapenem Hydrolysis very slow in aqueous solution. k cat =300 s -1, K m =166  M for CphA. Lactam ring opening

12. Validity of SCC-DFTB B3LYP/6-31G* (SCC-DFTB) [Experiment]

13. Proposed mechanism Garau et al. J. Mol. Biol. (2005)

14. Aims What is the substrate binding configuration? Can the non-metal-binding water serve as the nucleophile? Where is the general base? Is proton transfer concerted with nucleophilic addition? What is the role of metal? Is there tetrahedral intermediate?

15. Computational details Biapenem manually docked into active site. Stochastic boundary conditions. 500ps MD with 1fs time step and SHAKE. PMF from umbrella sampling + WHAM. B3LYP/6-31G(d,p) for truncated active-site model.

16. Active-sites (QM/MM simulations) Apo enzymeMichaelis complex His118 Asp120 Cys221 His263 Lys224 Asn233 Zn ++ Water11 Asn233 CO 3 2- Asp120 Lys224 His263 Cys221

17. Interaction pattern 500 ps QM/MM MD simulation Xu et al. J. Med. Chem., 2005 SCC-DFTB

18. Role of Asn233 in binding (X-ray) Zn 2+ Biapenem intermediate Asn233 (apo enzyme) Asn233 (enzyme- intermediate complex)

19. Experiment-theory agreement Distance (Å) and Angle (deg.) Apo CphA enzymeCphA-biapenem complex QM/MM MDDFTExp.QM/MM MDDFTExp.* N 4 ···Zn 2+ ---3.38±0.583.712.22 O 1 /O 13 ···Zn 2+ 2.15±0.082.082.102.12±0.072.032.39 Zn 2+ ··· N ε2 (His263)2.04±0.062.152.052.04±0.072.072.13 Zn 2+ ··· O δ2 (Asp120)2.14±0.071.981.962.14±0.071.982.03 Zn 2+ ··· S(Cys221)2.31±0.062.292.192.34±0.082.342.27 O w ···C 7 ---3.54±0.583.21- O 3 /O 12 ···H ζ2 (Lys224)1.63±0.131.03 + -1.71±0.121.90- O 14 ···H d22 (Asn233)---2.05±0.26-- O 12 ···H-N(Asn233)---2.25±0.49-- O 13 ···H e2 (His196)---2.16±0.40-- C 7 -N 4 -C 3 ---124.4±6.4127.7- C 2 -S-C 17 ---106.9±3.5101.7105.4 O 1 /O 13 ···Zn 2+ ···O δ2 (Asp120)102.6±8.6101.0102.1124.2±11.6117.3161.8 O 1 /O 13 ···Zn 2+ ···S - (Cys221)112.0±8.0123.2120.4118.3±9.7102.496.3 O 1 /O 13 ···Zn 2+ ···N ε2 (His263)100.1±12.993.2101.095.5±5.098.481.6

20. Potential of mean force Xu et al. J. Biol. Chem, 2006

21. Ground state

22. Transition state

23. Enzyme-intermediate complex

24. Truncated active-site model E-S TS 35 kcal/mol PC E-I 3 kcal/mol B3LYP/6-31G**

25. Proposed mechanism

26. Summary for CphA Biapenem binds directly with Zn, in addition to a network of H-bonds. Non-metal-binding water serves as the nucleophile. A single transition state features concerted nucleophilic addition and proton transfer. Asp120 serves as the general base. Metal serves as an electrophilic catalyst. SCC-DFTB/CHARMM and DFT models agree.

27. L1 from S. maltophilia B3 subclass. Found in opportunistic pathogen. Broad substrate spectrum. Active with one or two zinc cofactors. Structures of apo enzyme and complex with a hydrolysis product available.

28. Aims What is the substrate binding configuration? What are the roles of the two metal cofactors, Zn1 and Zn2? Is the general base necessary? Is proton transfer concerted with nucleophilic addition? Is there tetrahedral intermediate?

29. Active site (QM/MM simulation) Xu et al. to be published

30. Interaction pattern

32. Reaction path

34. Potential of mean force  G ‡ =7.8 kcal/mol

35. DFT model (B3LYP/6-31G*)  G ‡ =22 kcal/mol

36. Proposed mechanism Michaelis complex Transition state Negative ion intermediate (observed in nitrocefin hydrolysis by B. fragilis, Benkovic, 1998)

37. Summary for L1 Addition of OH - nucleophile is concerted with elimination of leaving group, with no tetrahedral intermediate. Proton transfer to Asp120 is delayed. Zn1 serves as oxyanion hole, while Zn2 stabilizes the anionic N leaving group. SCC-DFTB/CHARMM and DFT models are consistent.

38. Conclusions SCC-DFTB/MM approach is efficient and reasonably accurate, particularly in describing geometries. SCC-DFTB/MM approach gives qualitatively correct reaction mechanism, but might be off quantitatively. Reaction path and PMF reveal catalysis mechanisms in metallo-  -lactamases.

39. Acknowledgements National Institutes of Health (NIAID) National Science Foundation (MCB, CHE) National Center for Supercomputer Applications Prof. Q. Cui (U. Wisconsin) Prof. D. Xie (Nanjing U, China)