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)