Presented by: Seth Cory and Trang Nguyen TAMU Fall 2014

Acetyl-coenzyme A Synthase: A Novel Organometallic Catalyst and Drug Target

A Cluster: active site of ACS reaction

Proximal Ni: (trigonal planar) + where substrate binds + very labile can be removed by phenanthroline

Proximal Ni: (trigonal pyramid) can be replaced by Zn and Cu deactivates ACS

Ni

Lindahl, P. Coordination & Bioinorganic Chemistry Lectures, Nickel Enzyme, Texas A&M University, College Station. TX, USA, 2014

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Macharak, P; Harroop, T. Coord. Chem. Rev. 2005, 249, 3007-3024. Crabtree, R. The Organometallic Chemistry of the Transition Metals. 2005.

Active site of C-cluster Proposed Mechanism of C-cluster

How CO is delivered from C-cluster to A-cluster?

ACS/CODH Overview of Reaction

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BUT… the assembly chemistry of ACS is still unknown!!!

Darnault, C, et al. Nat. Struct. Biol. 2003, 10, 271-278.

Structure of ACS/CODH

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diffusion

Corrinoid Fe4S4 protein

????

Closed conformation Open conformation

What is the interface domain between CoFeSP and A cluster?

ACS/CODH Overview of Reaction

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Understand the chemical mechanism of A-cluster

Ni2+N

S N

S

O

Ni0S

2+/1+[Fe4S4]

Ni2+N

S N

S

O

Ni2+S2+/1+[Fe4S4]

Ni2+N

S N

S

O

Ni2+

S2+/1+[Fe4S4]

Ni2+N

S N

S

O

Ni2+

S2+/1+[Fe4S4]

CO

CH3Co3+-CoFeSP Co1+-CoFeSP

CO

H3C

Migratory InsertionH3C

O

CoAS-

H3C S

O

CoA

H3C

Dimagnetic Mechanism

Step 1: Oxidative elimination with CH3

- (Ni0 Ni2+) Step 2:

CO association (Ni2+)

Why do we study these interactions?

Adapted from: Lindahl, P. Met. Ions Life Sci. 2009, 6, 133-150.

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Understand the chemical mechanism of A-cluster

Ni2+N

S N

S

O

Ni1+S2+[Fe4S4]

Ni2+N

S N

S

O

Ni1+S2+[Fe4S4]

Ni2+N

S N

S

O

Ni2+

S2+[Fe4S4]

Ni2+N

S N

S

O

Ni3+S

2+[Fe4S4]

CO

CH3Co3+-CoFeSP

Co1+-CoFeSP

CO

CO

H3CMigratory Insertion

H3C

O

CoAS-

H3C

S

O

CoA

Ni2+ is activated by a 1e-

reduction by ferredoxin

Ni2+N

S N

S

O

Ni2+S

2+[Fe4S4]

CO

H3C

Internal e- transfer

Paramagnetic Mechanism

Step 1: CO association (Ni1+)

Step 2: Oxidative elimination

with CH3-

(Ni1+ Ni3+)

Why do we study these interactions?

Adapted from: Ragsdale, S; et. al. Chem. Rev. 2014, 114, 4149-4174.

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Why do we study these interactions?

Understand the chemical mechanism of A-cluster

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Monsato Acetic Acid Acetyl CoA Synthase

OXIDATIVE ADDITION with methyl group is a rate-limiting step

Temperature: 150-200 oC Temperature: 60-65 oC Pressure: 30-60 bar Pressure: 1 bar

How can we find the answer????

Lindahl, P. Coordination & Bioinorganic Chemistry Lectures, Nickel Enzyme, Texas A&M University, College Station. TX, USA, 2014

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Why do we study these interactions?

Understand the chemical mechanism of A-cluster

Develop novel catalysts in industrial reactions

TAMU CHEM 462

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Aim 3: To study the dynamics ACS/CODH enzyme

Aim 2: To probe coordination sphere of the A cluster

Aim 1: To study the methyl transfer from CoFeSP to ACS active site

Proposals for studying Acetyl CoA Synthase

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How can we study these interactions?

Ando, N.,et al. J. Am. Chem. Soc. 2012, 134, 17945–17954.

Darnault, C, et al. Nat. Struct. Biol. 2003, 10, 271-278.

Biophysical Techniques to study the interactions

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Ribonucleotide Reductases: A Model Study

Small Angle

X-ray Scattering

Experimental solution

scattering

Electron

Microscopy

X-ray

crystallography

Ando, N; et al. Proc. Natl. Acad. Sci. USA. 2011, 108, 21046-21051.

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Small Angle Xray Scattering

Molecular Envelopes

Electron Microscopy

Barondeau, D. Physcial Methods in Biological Chemistry, Structural Techniques, Texas A&M University, College Station. TX, USA, 2013

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X-ray Crystallography

Barondeau, D. Physcial Methods in Biological Chemistry, Structural Techniques, Texas A&M University, College Station. TX, USA, 2013

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How can we study these interactions?

Electron Microscopy (EM)

Small Angle X-ray Scattering (SAXS)

X-ray Crystallography (XTAL)

Ando, N.,et al. J. Am. Chem. Soc. 2012, 134, 17945–17954.

Darnault, C, et al. Nat. Struct. Biol. 2003, 10, 271-278.

Biophysical Techniques to study the interactions

Structural insight

at the interface

of CoFeSP & ACS

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How can we study these interactions?

Ando, N.,et al. J. Am. Chem. Soc. 2012, 134, 17945–17954.

Darnault, C, et al. Nat. Struct. Biol. 2003, 10, 271-278.

Docking Model between CoFeSP & ACS/CODH

Site-directed Mutagenesis at the interface

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Aim 3: To study the dynamics ACS/CODH enzyme

Aim 2: To probe coordination sphere of the A cluster

Aim 1: To study the methyl transfer from CoFeSP to ACS active site

Proposals for studying Acetyl CoA Synthase

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Site-directed Mutagenesis of A-cluster

Fontecilla-Camps, Handbook of Metalloproteins. 397-412

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Design primers with site-directed mutagenesis

Polymerase Chain Reaction (PCR)

DpnI Digestion & Transformation to E.coli cells

DNA sequencing to confirm the site-directed mutagenesis

Grow cells in large scale to obtain cell lysates

PURIFY the proteins (variants)

Site-directed Mutagenesis Procedure

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2. Kinetics with Crystallography

Site-directed Mutagenesis of A-cluster

1. Functional Assays: Acetyltransferase activity Acetyl CoA synthesis reaction rate

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𝛂 subunit 𝛂 subunit 𝛃 subunit

𝐀𝐀𝐀 𝐀𝐀𝐀 𝐀𝐂𝐂𝐂 Open conformation Tunnel is obstructed NO binding of CO

Closed conformation Tunnel is open Binding of CO

CO

What are the dynamics of the protein?

Structure of ACS/CODH

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Aim 3: To study the dynamics of ACS/CODH enzyme

Aim 2: To probe coordination sphere of the A cluster

Aim 1: To study the methyl transfer from CoFeSP to ACS active site

Proposals for studying Acetyl CoA Synthase

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Docking Model between ACS & CoFeSP

Predict the binding domain between CoFeSP & ACS/CODH

Strategy: Fluorescence Labeling at Specific Residues

a) Fluorescence Resonance Energy Transfer

A A

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Docking Model between CoFeSP & ACS/CODH

Predict the binding domain between CoFeSP & ACS/CODH

Strategy : Fluorescence Labeling at Specific Residues

a) Fluorescence Resonance Energy Transfer

b) Fluorescence Anisotropy

27 Fluorescence Resonance Energy Transfer

Fluorescence Anisotropy

Experimental Techniques to study:

Open conformation Closed conformation

Docking Model between CoFeSP & ACS/CODH

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Understand the methyl transfer from CoFeSP to ACS

Understand the dynamics of ACS

Understand

the chemistry

of ACS

Conclusion

Signs & Symptoms: Severe diarrhea Colitis Toxic megacolon Intestinal perforations

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How serious is CDI?

McCollum DL, Rodríguez M. Clin. Gastroenterol. Hepatol , 2012, 10, 581-592.

Current Treatments: Vancomycin: inhibitor of bacterial cell wall synthesis Metronidazole: inhibitor of bacterial DNA synthesis Antibiotics

Resistance A drive to find new target for the treatment of CDI

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Acetyl CoA Synthase: a new target for CDI treatment

Zhu, X. et al. Metallo., 2013, 5, 551-558.

3 potent inhibitors to inactivate ACS potent to C.difficile: 8-hydroxyquinoline 1,10-phenanthroline 2,2-dipyridyl

Inhibit methyl transfer

New antibiotics for CDI treatment

Understanding the chemistry of ACS is important to the following fields: Bioinorganic Chemistry Biomimetic/Organometallic Catalysis Medicinal Chemistry

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Overview

McCollum DL, Rodríguez M. Clin. Gastroenterol. Hepatol , 2012, 10, 581-592.

Several biophysical and biochemical studies need to be done TEM, SAXS, X-ray crystallography Site-directed mutagenesis Fluorescent studies