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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. Metallo- b -lactamases. One of four classes (B) of bacterial hydrolases responsible for penicillin resistance. - PowerPoint PPT Presentation
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Binding and Catalysis of Metallo--Lactamases Studied using a SCC-
DFTB/Charmm Approach
D. Xu and H. GuoDepartment of ChemistryUniversity of New Mexico
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
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.
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.
Compromise: QM/MM method
QM potential for reaction region.
MM force field for surrounding and solvent.
Boundary.
MM
QM
Enzyme
Substrate
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.
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
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.
Hydrolysis of biapenem
Hydrolysis very slow in aqueous solution. kcat=300 s-1 , Km=166 M for CphA.
Lactam ring opening
Validity of SCC-DFTB
B3LYP/6-31G*(SCC-DFTB)[Experiment]
Proposed mechanism
Garau et al. J. Mol. Biol. (2005)
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?
Active-sites (QM/MM simulations)
Apo enzyme Michaelis complex
His118
Asp120Cys221 His263
Lys224
Asn233
Zn++Water11
Asn233
CO32-
Asp120 Lys224His263
Cys221
Interaction pattern
500 ps QM/MM MD simulationXu et al. J. Med. Chem., 2005
SCC-DFTB
Experiment-theory agreement
Distance (Å) andAngle (deg.)
Apo CphA enzyme CphA-biapenem complex
QM/MM MD DFT Exp. QM/MM MD DFT Exp.*
N4···Zn2+ - - - 3.38±0.58 3.71 2.22
O1/O13···Zn2+ 2.15±0.08 2.08 2.10 2.12±0.07 2.03 2.39
Zn2+··· Nε2(His263) 2.04±0.06 2.15 2.05 2.04±0.07 2.07 2.13
Zn2+··· Oδ2(Asp120) 2.14±0.07 1.98 1.96 2.14±0.07 1.98 2.03
Zn2+··· S(Cys221) 2.31±0.06 2.29 2.19 2.34±0.08 2.34 2.27Ow···C7 - - - 3.54±0.58 3.21 -
O3/O12···Hζ2(Lys224) 1.63±0.13 1.03+ - 1.71±0.12 1.90 -
O14···Hd22(Asn233) - - - 2.05±0.26 - -
O12···H-N(Asn233) - - - 2.25±0.49 - -
O13···He2(His196) - - - 2.16±0.40 - -
C7-N4-C3 - - - 124.4±6.4 127.7 -
C2-S-C17 - - - 106.9±3.5 101.7 105.4
O1/O13···Zn2+···Oδ2(Asp120) 102.6±8.6 101.0 102.1 124.2±11.6 117.3 161.8
O1/O13···Zn2+···S-(Cys221) 112.0±8.0 123.2 120.4 118.3±9.7 102.4 96.3
O1/O13···Zn2+···Nε2(His263) 100.1±12.9 93.2 101.0 95.5±5.0 98.4 81.6
Potential of mean force
Xu et al. J. Biol. Chem, 2006
Ground state
Transition state
Enzyme-intermediate complex
Truncated active-site model
E-S
TS 35 kcal/mol
E-I3 kcal/mol
B3LYP/6-31G**
Proposed mechanism
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.
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.
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?
Active site (QM/MM simulation)
Xu et al. to be published
Interaction pattern
Interaction pattern
Reaction path
Reaction path
Potential of mean force
G‡=7.8 kcal/mol
DFT model (B3LYP/6-31G*)
G‡=22 kcal/mol
Proposed mechanism
Michaelis complex Transition state Negative ion intermediate (observed in nitrocefin
hydrolysis by B. fragilis, Benkovic, 1998)
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.
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.
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)