Sandbox 156: Difference between revisions

Chloramphenicol acetyltransferase type III (CAT III) is an enzyme which catalyzes the transfer of the acetyl group from [http://en.wikipedia.org/wiki/Acetyl-CoA acetyl-CoA] to hydroxyl groups of [http://en.wikipedia.org/wiki/Chloramphenicol chloramphenicol]. CAT III is a trimeric protein with a ''Mr'' of 25 000-kDa and a member of the [http://en.wikipedia.org/wiki/Acetyltransferase actetyltransferase family] of proteins.

[http://en.wikipedia.org/wiki/Peptidyl_transferase peptidyl transferase] activity<ref name=”Day”>PMID: 1544895</ref>. However, when CAT III catalyzes the acetylation of chloramphenicol, the antibiotic can no longer bind to the ribosomes and is rendered inactive. The genes for the enzyme are commonly found on the plasmid of the bacteria and have been found in a numerous bacterial species<ref>PMID: 2268277</ref>.

The multifunctional enzyme consists of three identical subunits with three active sites at the subunit interfaces. The side chains of one subunit allow [http://en.wikipedia.org/wiki/Van_der_Waals_force van der Waals interactions] and two hydrogen bonds with chloramphenicol, causing binding of the substrate. The opposing subunit provides a histidine (His-195) residue essential for catalysis<ref>PMID: 8407936</ref>. Water molecules in the cavity provide a bridging hydrogen bond between the 1-hydroxyl of chloramphenicol and the hydroxyl of a threonine (Thr-174) residue. The active site of CAT III performs two acetylations of chloramphenicol and can accommodate the presence of the first intermediates quite well<ref name="Murray">PMID:2015231</ref>.

Both reactions take place in the active site of CAT III, where acetyl-CoA is tunneled through from the opposing side of the trimer<ref>PMID: 3288984</ref>.

[[Image:Picture2.jpg|500x400 px|center]]

In the first step of the reaction, Histidine-195 abstracts a proton from the 3-hydroxyl of chloramphenicol, promoting a [http://en.wikipedia.org/wiki/Nucleophilic_attack nucleophilic attack] from the [http://en.wikipedia.org/wiki/Oxyanion oxyanion] to the thioester bond of the acetyl-CoA. The intermediate produced, 3-acetylchloramphenicol, then rearranges non-enzymatically to 1-acetylchloramphenicol. Regeneration of the 3-hydroxyl allows another round of CAT III catalyzed nucleophilic attack and a 1,3-diacetylchloramphenicol product is formed<ref name="Murray"/>.

[[Image:512px-Acetyl-CoA-2D.svg.png|thumb|300x1000 px|right|Structure of Acetyl-CoA]]

The general structure of CAT III is dominated by a six stranded antiparallel <scene name='Sandbox_156/Scene_4/4'>β-sheet</scene> and 5 <scene name='Sandbox_156/Scene_4/3'>α-helices</scene> stacked against the ends and face of the protein, forming a structure known as an "open-faced sandwhich"<ref>PMID: 3288984</ref>. An extended β-strand forms an extension of the six stranded sheet to a seven stranded sheet that spans the interface of the subunit.  Three identical monomers associate to form the trimeric protein with two <scene name='Sandbox_156/Scene_7/1'>cobalt</scene> ions acting as [http://en.wikipedia.org/wiki/Cofactor_%28biochemistry%29 cofactors].

The association of the monomeric subunits produces a well-defined pocket at the subunit interface, allowing for the binding of the chloramphenicol molecule. The <scene name='Sandbox_156/Scene_3/1'>chloramphenicol binding site</scene> of CAT III is lined with hydrophobic residues, allowing only 2 hydrogen bonds with the substrate. A third hydrogen bond is mediated through a <scene name='Sandbox_156/Scene_4/2'>bridging water molecule</scene> and allows interaction between the the 1-hydroxyl of chloramphenicol and the hydroxyl of Tyr-174<ref name="Murray"/>.

The binding of chloramphenicol blocks acetyl-CoA from entering the active site from the <scene name='Sandbox_156/Scene9/1'>top of the enzyme</scene>, as illustrated. Instead, the second substrate tunnels to the active site through the  

<scene name='Sandbox_156/Scene9/2'>pantetheine arm</scene> from the opposite side of the trimer. The amino acids lining the tunnel are also primarily lined with hydrophobic residues allowing for close contact between the substrate and aromatic residues. This tunneling allows the formation of van der Waals interactions between the adenine ring of acetyl-CoA with dimethyl groups of the pantetheine arm and hydrogen bonding of the ring with Tyr-56,Phe-96 and Phe-103, binding the substrate.  

The active site catalyzes sequential acetylations of the chloramphenicol molecule. This is initiated by the deprotonation of the 3-hydroxyl of chloramphenicol by <scene name='Sandbox_156/Scene_two/1'>His-195</scene><ref name="Murray"/>. Numerous hydrogen bonds are present and, importantly, van der Waals interactions between the benzene ring of the <scene name='Sandbox_156/Scene_4/1'>Tyr-25</scene> residue and the imidazole ring. This van der Waals contact may help stabilize side-chain orientations, promoting specificity in the reaction. The acetyl group is already properly positioned after tunneling, and reacts readily without any major structural changes<ref>PMID: 3288984</ref>.