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== '''Bifunctional Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase (CODH/ACS)''' ==
<Structure load='1mjg' size='400' frame='true' align='right' caption='Carbon monoxide dehydrogenase/acetyl-CoA synthase asymmetric unit containing two α2β2 tetramers.' scene='Insert optional scene name here' />


 
=== '''Introduction''' ===
'''Cholix Toxin''' named also CT, is a protein toxin that uses ADP-ribosyltransferases to modify target proteins in human cells. The mono-ADP-ribosyltransferase toxins are bacterial virulence factors that are believed to be a factor in many disease states in animals plants as well even humans. For a list of various and other similar toxins see [[Toxins]].
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Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a bifunctional protein which acts as both an [http://en.wikipedia.org/wiki/Oxidoreductase oxidoreductase] and a [http://en.wikipedia.org/wiki/Transferase transferase] by reducing carbon dioxide to carbon monoxide (or the reverse oxidation of carbon monoxide to carbon dioxide) and then catalyzing the synthesis of [http://en.wikipedia.org/wiki/Acetyl-CoA acetyl-CoA] from carbon monoxide, coenzyme A, and the methyl group of an corrinoid iron-sulfur protein.  This enzyme plays a key role in the Wood-Ljungdahl pathway which is used by anaerobic, [http://en.wikipedia.org/wiki/Autotroph autotrophic] bacteria such as ''Moorella thermoacetica'' (f. ''Clostridium thermoaceticum'') and ''Clostridium ljungdahlii'' for gaseous carbon fixation. End products of the Wood-Ljungdahl pathway include cell biomass, acids (ex. acetate and butyrate), and alcohols (ex. ethanol and butanol) – all of which derive from acetyl-CoA. The Wood-Ljungdahl pathway is also the most energetically favorable carbon fixation pathway, but its use is confined to only obligate, anaerobic species <ref name="Berg">PMID:20453874</ref>.  Since the discovery of the Wood-Ljungdahl pathway in the early 1980’s, significant effort has been put into trying to characterize the substrate binding activity of CODH/ACS with the ''M. thermoacetica'' protein used as the model in most case studies <ref name="CN">PMID:19583207</ref>.  However, little progress was made in defining the exact structure of the protein with crystal structures until the 2000’s. Several reasons why CODH/ACS has received so much attention include the fact that it contains highly disputed metalloclusters, its use of biological organometallic intermediates in reactions, and its contribution to reducing environmental pollutants <ref>PMID: 11841199</ref> <ref>PMID: 11848835</ref>.  
<Structure load='3ki4' size='300' color='white' frame='true' align='right' caption='X-Ray Diffraction image at 2.1Å of Cholix Toxin from Vibrio Cholerae' />
 
 
 
 
== Cholix Toxin from ''Vibrio Cholerae ''==
The [http://en.wikipedia.org/wiki/Crystal_structure crystal structure] of the purified form of ''' Cholix Toxin''' or '''CT''' was determined in 1995.It is an oligomeric bacterial protein found to be made up of six individual subunits, one single α-subunit and 5 individual β- subunits.The α-subunit makes up what is known as the enzymatic portion of the protein while the 5 copies of the β-subunit are responsible for the binding to the ligand receptor. The toxin binds highly specifically and tightly to a [http://en.wikipedia.org/wiki/GM1_gangliosidoses GM1 gangliosides] on the surface of the host's cells. In this X-Ray Diffraction image we can see the <scene name='Sandbox_Reserved_496/Binding_site/1'>catalytic</scene> site, which in this case has been complexed with an allosteric inhibitor (red and yellow space filling atoms). Recent studies have indicated several amino acid residues located proximally to the active site which are critical for enzymatic activity. Specifically, site directed mutagenesis indicated that when altered, the mutation results in a termination of the proteins toxicity, rendering it essentially harmless.  




Similar bacteria which harbor monofunctional CODH enzymes include the autotrophs ''Rhodosprillum rubrum'' and ''Carboxydothermus  hydrogenoformans'' <ref name="Cu">PMID:12386327</ref>.  In addition, a variation on CODH/ACS termed CODH-containing acetyl-CoA decarboxylase/synthase is found in methanogenic  archea and decomposes acetyl-CoA to carbon dioxide and methane <ref>PMID: 18621675</ref>.




PDB codes for the ''M. thermoacetica'' CODH/ACS enzyme are: 1MJG <ref>1MJG. [http://dx.doi.org/10.2210/pdb1mjg/pdb DOI:10.2210/pdb1mjg/pdb]</ref>  <scene name='Sandbox_Reserved_496/Codh_acs/1'>(orginial scene)</scene>, 1OAO <ref>1OAO. [http://dx.doi.org/10.2210/pdb1oao/pdb DOI:10.2210/pdb1oao/pdb]</ref>, 2Z8Y <ref>2Z8Y. [http://dx.doi.org/10.2210/pdb2z8y/pdb DOI:10.2210/pdb2z8y/pdb]</ref>, 3I01 <ref>3I01. [http://dx.doi.org/10.2210/pdb3i01/pdb DOI:10.2210/pdb3i01/pdb]</ref>, and 3I04 <ref>3I04. [http://dx.doi.org/10.2210/pdb3i04/pdb DOI:10.2210/pdb3i04/pdb]</ref>.




==='''Structure'''===
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The CODH/ACS enzyme from ''M. thermoacetica'' is an <scene name='Sandbox_Reserved_496/Codh_acs_tetramer/2'>α2β2 tetramer</scene> with seven metalloclusters. Each 674 residue <scene name='Sandbox_Reserved_496/Beta_subunits/2'>β subunit </scene>  carries out CODH activity, while each 729 residue <scene name='Sandbox_Reserved_496/Alpha_subunits/1'>α subunit</scene> is responsible for ACS activity.  From the N-terminus to C-terminus, <scene name='Sandbox_Reserved_496/Beta_secondary/1'> β subunit domains</scene> are as follows: an α-helical domain (residues 1-257) followed by two α/β Rossmann-like domains (residues 262-458 and 463-674). The β subunit has 57% helical and 9% β-sheet character with 31 helices and 15 β-strands. The  <scene name='Sandbox_Reserved_496/Alpha_secondary/1'>α subunit is also comprised of three domains</scene>, two with α+β folds and a third with a helical region (residues 1-154) at the N-terminus of a Rossmann (six-stranded α/β) fold (residues 155-316) which is similar to a portion of the β subunit structure <ref name="Cu"/> <ref name="Xe">PMID:18293927</ref>.  Overall, the α subunit has 50% helical and 14% β-sheet character with 36 helices and 22 β-strands.




<scene name='Sandbox_Reserved_496/Beta_metalloclusters/1'>Five of the metalloclusters</scene> (<font color='red'>'''two B-'''</font>, <font color='blue'>'''two C-'''</font>, and <font color='green'>'''one D-cluster'''</font>) are located in the β<sub>2</sub> portion of the protein with a B- and C-cluster in each β subunit and the sole D-cluster bridging the two subunits.  The B- and D-clusters consist of [Fe<sub>4</sub>S<sub>4</sub>] centers that serve to transfer electrons.  As the active site in each β subunit where CODH activity occurs, the <scene name='Sandbox_Reserved_496/Beta_c_cluster/2'>C-clusters are composed of a Fe-[NiFe3S4] cage </scene> with the unique Fe coordinated by a bridging sulfur ion, His283, and Cys317 <ref name="Zn">PMID:12627225</ref>.  The B- and D-clusters are positioned in relation to the α-helical domain of the β subunit, while the C-clusters are aligned through interactions with both Rossmann-like domains <ref name="Xe"/>.  A ligand binding study with the native CODH/ACS structure and with cyanide used as competitive inhibitor for CO showed that H<sub>2</sub>O/OH<sup>-</sup> binds the C-cluster at the unique Fe and CO binds the C-cluster at the Ni ion <ref name="CN"/>.




The remaining two metalloclusters (both A-clusters) are located in the two α subunits of the CODH/ACS protein.  As the active site of ACS activity in each α subunit, the A-clusters are composed of a [Fe<sub>4</sub>S<sub>4</sub>] center bridged to a binuclear site.  There is some debate regarding the metal ions present in the binuclear site; either <scene name='Sandbox_Reserved_496/Alpha_a_cluster/1'>both A-clusters are [Fe4S4]-Cu-Ni</scene> <ref name="Cu"/> or one A-cluster is <scene name='Sandbox_Reserved_496/A_cluster_ni_ni/1'>[Fe4S4]-Ni-Ni</scene>  while the other is <scene name='Sandbox_Reserved_496/A_cluster_zn_ni/1'>[Fe4S4]-Zn-Ni</scene>  <ref name="Zn"/>.  The [Fe<sub>4</sub>S<sub>4</sub>] center is coordinated by Cys506, Cys509, Cys518, and Cys528 with Cys509 also forming the bridge between the [Fe<sub>4</sub>S<sub>4</sub>] and the [http://en.wikipedia.org/wiki/Proximal#Proximal_and_distal proximal] metal ion.  Furthermore, the distal metal ion is coordinated by Cys595, Gly596, and Cys597 with Cys595 and Cys597 also connecting the distal and the proximal metal ions.  Only the [Fe<sub>4</sub>S<sub>4</sub>]-Ni-Ni  A-cluster is in an open conformation, the other two site types with Cu or Zn as the proximal ion are in closed form.     




Another characteristic feature of CODH/ACS is a 138Å long  <scene name='Sandbox_Reserved_496/Codh_acs_hydrophilic/1'>hydrophobic tunnel</scene> that connects the C- and A-clusters.  The cavity network formed by the hydrophobic tunnel is an S-shaped pore from C-cluster to C-cluster with two long branches that lead from the “S” to each A-cluster.  As the tunnel runs along nearly the full length of the protein, it enables the intramolecular diffusion of CO from the CODH active sites to the ACS active sites <ref name="Cu"/>. 




X-ray crystallography methods were used to determine the structure of CODH/ACS from ''M. thermoacetica''.  Crystals were formed by means of sitting drop vapor diffusion.  Both molecular replacement (using the CODH structure of ''R. rubrum'' <ref name="Cu"/> and ''C. hydrogenoformans'' <ref name="Zn"/>) and multiple-wavelength anomalous dispersion (MAD) methods were used to solve the structure’s diffraction pattern.  Resolution of the structures at the highest resolution shell ranges from 2.5Å to 1.9Å.


==='''Mechanism of Action'''===
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[[Image:A_cluster_1.JPG|frame|Proposed mechanism  1 for ACS activity with Cu-Ni ions in the binuclear site of the A-cluster. <ref name="Cu"/>]]


== '''Toxin Mechanism''' == 
'''β subunit reactions'''
Once secreted, the B subunit will bind to GM1 gangliosides on the surface. After binding takes place, the whole complex is engulfed by the cell and a portion known as the CTA1 chain is detached after reduction of a disulfide bridge. The new endosome is moved to the Golgi, where it is recognized by the endoplasmic reticulum, unfolded and delivered to the membrane, where the Endoplasmic Reticulum-oxidase - "'''Ero1'''" triggers the release of the excised A1 protein (through Oxidation) of '''protein disulfide isomerase complex'''. As A1 moves from the ER into the cytoplasm it refolds and avoids further reduction.[1]


C-cluster (CODH activity): CO<sub>2</sub> + 2H<sup>+</sup> + 2e<sup>-</sup>  ↔  CO + H<sub>2</sub>O


The A1 fragment catalyses '''ADP ribosylation''' from NAD to the regulatory component (G-protein) of adenylate cyclase, two main components in an important signal transduction pathway. The newly formed A1-Gαs complex is then unable to hydrolyse properly leaving the GTP bound to the Gαs subunit, which results in the transducer being continually activated. Increased adenylate cyclase activity increases cyclic AMP (cAMP concentration increases more than 100 times normal concentrations) synthesis. This can cause rapid fluid loss from the intestines, up to 2 liters per hour, leading to severe dehydration and diarrhea.
B- and D-clusters: electron transfer




'''α subunit reactions'''


[[Image:Cholera_Mechanism.jpg]]
A-cluster (ACS activity): CH<sub>3</sub>-Co(III)-CFeSP + CO + HSCoA  ↔  CH<sub>3</sub>-CO-SCoA +  Co(I)-CFeSP + H<sup>+</sup>




There are two proposed mechanisms for the catalytic action of the A-cluster:  mechanism 1 uses Cu-Ni ions in the binuclear site while mechanism 2 uses Ni-Ni ions. 


Mechanism 1 suggests that CO created at the C-cluster travels through the hydrophobic tunnel and binds to Cu.  Next, the A-cluster switches to an open corrinoid conformation so that the methyl group may be transferred from the corrinoid iron-sulfur protein to the distal Ni ion of the active site.  The cluster reverts back to a solvent protected, closed form to generate the acetyl intermediate, and then reopens to allow HSCoA to bind.  HSCoA likely binds in the large cavity between the three domains of the α subunit where six Arg residues and Trp418 coincide.  Finally, HSCoA is deprotonated and acetylated to form acetyl-CoA <ref name="Cu"/>.
{{clear}}


[[Image:A_cluster_2.JPG|frame|Proposed mechanism  2 for ACS activity with Ni-Ni ions in the binuclear site of the A-cluster. <ref name="Zn"/>]]




Mechanism 2 proposes that CO binds to the proximal Ni ion after exiting the tunnel.  The A-cluster then changes from the closed to the open conformation which closes the hydrophobic CO tunnel and provides a site for methyl group binding on the proximal Ni as it transitions from Ni(0) to Ni(II).  Next, the bound CO inserts into the Ni-CH<sub>3</sub> bond to produce an acetyl intermediate.  Lastly, deprotonated CoA-S<sup>-</sup> attacks the carbonyl carbon of the acetyl group to produce acetyl-CoA and the proximal Ni is reduced back to Ni(0) <ref name="Zn"/>.  Note that this mechanism differs from the former by binding both CO and CH<sub>3</sub> to the proximal metal ion and also the A-cluster stays open between methylation and CoA acetylation.
{{clear}}


[[Image:C_cluster.JPG|frame|Proposed mechanism for CODH activity in the C-cluster. <ref name="CN"/>]]


In the proposed catalytic action of the C-cluster, CO first binds the Ni ion followed by deprotonation of the Fe bound water to yield a reactive hydroxide which promotes nucleophilic attack of CO by the hydroxide.  Next, the Ni-COOH intermediate is deprotonated to Ni-COO<sup>-</sup> which allows release of CO<sub>2</sub> as the C-cluster is reduced to a new redox state.  Then, electrons are shuttled between the B- and D-clusters to reoxidize the C-cluster to its original redox state <ref name="CN"/>.  This catalytic cycle is of course operated in reverse to reduce CO<sub>2</sub> to CO and allow subsequent production of acetyl-CoA .


{{clear}}


==='''Possible Applications'''===
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Since CODH/ACS consumes CO<sub>2</sub> it plays a direct part in the reduction of greenhouse gases by converting CO<sub>2</sub> into an intermediate which can transformed into more desirable products such as alcohols and acids.  A novel application in which CODH/ACS plays a role is the production of bioethanol from renewable natural resources such as wood, grasses, and agricultural residues (ex. corn stover).  In this process, the biomass undergoes [http://en.wikipedia.org/wiki/Gasification gasification] to yield CO, CO<sub>2</sub>, and H<sub>2</sub>.  The gases are subsequently fed to bacterial reactor systems containing organisms which utilize the Wood-Ljungdahl pathway and thus produce ethanol.  The main goal is to produce a renewable energy source that may be used as an alternative to petroleum.  However, at this time, the flux of carbon through the Wood-Ljungdahl pathway does not tend to favor ethanol production so much work remains to be done in this area.  On the other hand, the Wood-Ljungdahl pathway does produce significant levels of acetic acid, and to this extent, the pathway is considered a biological equivalent to the [http://en.wikipedia.org/wiki/Monsanto_process Monsanto process] for industrial acetic acid production <ref name="Berg"/>.  Roughly 10% of the total biological acetic acid production is attributable to anaerobic, autotrophic bacteria which possess CODH/ACS enzymes.


== '''Uses & Potential Benefits''' ==
==='''References'''===
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<references/>
--[[User:Rachel Slivka|Rachel Slivka]]
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