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Project 1�DD-Peptidase/β-Lactamase-Catalyzed Reactions �with QM/MM��Hao Xu

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Objective

Ancient DD-Peptidase

Lys₁NH₃⁺

Ser₂/TyrOH

AsnCONH₂

Ser₁OH

HO-Ser/Thr

H3N+Lys₂

Gly

Antibiotics

R. F. Pratt, J. Med. Chem. 2016, 59, 8207−8220

Present DD-Peptidase

Present β-lactamase

Evolution

Present DD-Peptidase

Present β-lactamase

Deacylation

Acylation

Fast

Slow

Fast

Hydrolysis of β-Lactam Antibiotics

Active Site

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Material: DD-Peptidase (PDB code 2J8Y) β-Lactamase (PDB code 5KMW)	Approaches: Molecular Dynamics Simulation QM/MM; Replica Path Method
DD-Peptidase (PDB code 2J8Y)

Material and Approaches

-

+

Total QM/MM

MM

QM

MM

MD: determine the optimal conformation.

QM/MM: computing energy.

Soumillion, P. et al J. Mol. Biol. 2009, 386, 109–120; Brooks, B. R. et al J. Chem. Theory Comput. 2014, 10, 4198–4207

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Replica Path Method

Brooks, B. R. et al J. Chem. Theory Comput. 2014, 10, 4198–4207

Define replicas along the path as intermediate steps between the reactant and product. Replica = λ*R + (1- λ)*P
Optimize each replica with restraints to get the minimal energy reaction pathway.

Restraint Terms

Replica Core Szie (k_core): optimized region, size >= QM region(4 Å). (Å)

Distance Restraint(k_rms): force constant, used to restrain the distance between adjacent replicas to keep the path smooth and evenly spaced. (kcal/mol/Å²)

Angle Restraint(k_angle): force constant, used to restrain the angle between adjacent and next replicas. (kcal/mol/Å²)

The Replica Path Method is a useful tool for exploring reaction pathways. Given the structures of the reactant and product, intermediate replicas are defined along the pathway, each with a structure that combines portions of the reactant and product structures.

Next, the initial replicas are optimized using restraints, allowing us to obtain the minimal energy reaction pathway. Without restraints, the replicas would tend to converge to the global minimum or get trapped in a local minimum, resulting in an inadequate sampling of the reaction pathway. This, in turn, leads to an underestimation of the reaction energy barrier. Therefore, the use of restraints in the optimization process is crucial.

In the optimization step, only the replica core gets optimized. Its size has to be larger than QM region. Replica Core Szie is a very important restraint.

Distance Restraint is force constant, a 2^nd derivative of free energy, which is used to restrain the distance between adjacent replicas to keep the path smooth and evenly spaced.

Angle Restraint is also a force constant, which is used to restrain the angle between adjacent and next replicas.

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Proposed Mechanism

Active site

MD: Ser 61 stay close to Penicillin during the 10 ns simulation of DD-Peptidase.

Deacylation

Acylation

DD-Peptidase

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Parameter Optimization

k_rms

1000

2000

3000

5000

k_angle=1000, k_core= 4 Å

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Parameter Optimization

k_angle

100

1000

20000

50000

k_rms=5000, k_core= 4 Å

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Parameter Optimization

k_core

4 Å

6 Å

8 Å

10 Å

k_rms=5000, k_angle=100

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Reaction Pathway of Acylation

Optimal parameters: k_rms=5000, k_angle=100, k_core= 4 Å

DD-Peptidase

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Reaction Pathway of Deacylation

Optimal parameters: k_rms=5000, k_angle=100, k_core= 4 Å

DD-Peptidase

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Summary

Optimal parameters: k_rms=5000, k_angle=100, k_core= 4 Å

	Process	Activation Free Energy	Free Energy Difference
DD-Peptidase (2J8Y)	Acylation	18 kcal/mol	-3 kcal/mol
DD-Peptidase (2J8Y)	Deacylation	27 kcal/mol	+7 kcal/mol
β-lactamase (5KMW)	Acylation	14 kcal/mol	-2.1 kcal/mol
β-lactamase (5KMW)	Deacylation	8.7 kcal/mol	-12.9 kcal/mol

Present DD-Peptidase

Present β-lactamase

Deacylation

Acylation

Fast

Slow

Fast

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Acknowledgements

All former colleagues and classmates at SMU.

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