Binding and Catalysis of Metallo- b -Lactamases Studied using a SCC-DFTB/Charmm Approach

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

2. Metallo-b-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

3. 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.

4. 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.

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

6. 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.

7. SCC-DFTB Q. Cui, 2006 • 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.

8. 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.

9. Hydrolysis of biapenem • Hydrolysis very slow in aqueous solution. • kcat=300 s-1 , Km=166 mM for CphA. Lactam ring opening

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

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

12. 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?

13. Asn233 Asn233 CO32- His118 Zn++ Lys224 Water11 His263 Asp120 Lys224 Asp120 Cys221 Cys221 His263 Active-sites (QM/MM simulations) Apo enzyme Michaelis complex

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

15. Experiment-theory agreement

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

17. Ground state

18. Transition state

19. Enzyme-intermediate complex

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

21. Proposed mechanism

22. 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.

23. 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.

24. 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?

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

26. Interaction pattern

27. Interaction pattern

28. Reaction path

29. Reaction path

30. Potential of mean force DG‡=7.8 kcal/mol

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

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

33. 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.

34. 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-b-lactamases.

35. 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)