Thiolase_3_Ketoacyl_CoA

3-Ketoacyl-CoA Thiolase (PDB: 1AFW) Saccharomyces cerevisiae

Created by: Zach Davis

             3-Ketoacyl-CoA Thiolase (PDB: 1AFW) from Saccharomyces cerevisiae has the vital function of reversibly dividing 3-Ketoacyl-CoA into acyl-CoA and acetyl-CoA.  It is classified as Thiolase I.  All classes of Thiolases (I and II) have the ability to do this, but Thiolase I primarily cleaves 3-Ketoacyl-CoA and other fatty acids while Thiolase II synthesizes via a Claisen Condensation mechanism acetoacetyl-CoA from acyl-CoA and acetyl-CoA (1).    This process is a key component of the biosynthetic pathway of producing energy in the body (2).  The molecular weight of 3-Ketoacyl-CoA Thiolase is 44730.35 Da with an isoelectric point at pH of 7.52 (3).

3-Ketoacyl-CoA Thiolase is a peroxisomal enzyme studied in Saccharomyces cervisiae.  This organism is used because solely peroxisomal enzymes do all fatty acid β-oxidation in this organism including both saturated and unsaturated fats. This is important to humans because of the group of inheritable diseases identified in humans cause peroxisomal enzyme deficiency.  The peroxisomal enzymes are genetically conserved between yeast and humans which makes yeast the perfect organism to study the peroxisomal fatty acid β-oxidation and its effects.   3-Ketoacyl-CoA thiolase is the enzyme that carries out the final step in the β-oxidation cycle.  In this step, 3-ketoacyl-CoA esters are thiolytically cleaved into acyl-CoA and acetyl-CoA.  These molecules produce energy for the cell (7).

Acetyl-CoA, especially from Saccharomyces cerevisiae, is also used in the production of biofuels because it is a precursor to n-butanol.  In yeast, the production of Acetyl-CoA can be amplified to make it essentially a cellular factor for Acetyl-CoA and subsequent n-butanol.  Part of amplifying the production of this compound is the study of the peroxisomal enzymes β-oxidation of fatty acids into this Acetyl-CoA.  In other words by better understanding 3-Ketoacyl-CoA thiolase in yeast, more biofuel metabolites can be produced which would lead to the increased production of biofuels as a renewable energy source (11).

A protein’s structure is indicative of its function.  This protein has two subunits that interact with each other over a large portion of each dimer in order to form a deep pocket for the active site to trap 3-Ketoacyl-CoA. The two secondary structures present are regular βα-repeats.  41% of the protein is helical and 21% is beta sheets.   The helical portion has 22 helices and 163 residues.  The beta sheets have 21 strands and 83 residues (9).  The two central helixes are completely inside the dimer with the non-polar sides facing the inside of each dimer and the polar side facing the cavity in order to trap water molecules. These two dimers are most heavily associated at each subunit’s N-Domain.  Two salt bridges help connect the two dimers.  The two opposing sides of the pocket are hydrophobic which help to maintain the structure of the pocket.  Within this pocket, there are no charged side chains within 9 Å of the activation site.  This prevents any interference with the interacting between the 3-Ketoacyl-CoA and other non-activation site residues (1).

3-Ketoacyl-CoA thiolase exists as a homodimer although most are homotetramers.  From two of the most studied tetrameric thiolases—acetoacetyl-CoA thiolase from Zoogloea ramigera and mitochondrial Ketoacyl-CoA thiolase from a pig heart—two cysteine residues have been identified as the main catalytic components of the thiolases primary function (1)(4).  

In 3-Ketoacyl-CoA thiolase in S. cerevisiae, these are Cys-125 and Cys-403.  His-375 has also been predicted as a proton donor/acceptor in this reaction, and Gly-405 hydrogen bonds with carbonyl oxygen on the soon to be acyl group to hold the 3-Ketoacyl-CoA in place.  While Gly-405 is holding the 3-Ketoacyl-CoA in place in the active site, nucleophilic Cys-125 attacks the carbonyl carbon of the soon to be acyl group and cleaves the carbon-carbon bond.  After this occurs, His-375 donates a proton to nucleophilic Cys-403 in order to prevent the Cys-403 from nucleophilically attacking the remaining carbonyl carbon.  The acetyl-CoA leaves and CoASH enters the active site.   His-375 accepts a proton from CoASH in order to create the nucleophile CoAS-.  The CoAS- nucleophilically attacks the carbonyl carbon on the acyl group attached to Cys-125 and cleaves the acyl group from Cys-125.  This forms the product Acyl-CoA (1).  

3-Ketoacyl-CoA thiolase is similar in S. cerevisiae  to 3-Ketoacyl-CoA in Arabidopsis (2WU9).  They are similar in the function of the β-oxidation of fatty acids into acetyl-CoA.  Fatty acids are an important molecule in nature for both plants and animals. Because of the variety in which they occur—varying degrees of saturation and length—they are excellent energy storage molecules.  These fatty acids are broken down two carbons at a time. 

Because structure is so important to function in proteins, it stands to reason that the structure of 3-Ketoacyl-CoA thiolase in Arabidopsis would have a similar structure to 3-Ketoacyl-CoA thiolase in S. cerevisiae.   This turns out to be true with a Z-score of 61.8.  Z-score is a comparison of intermolecular distances within the tertiary structures.  This results in a quantitative measure of the folding similarity between the two proteins.  If a protein has a Z-score greater than two, then it is considered to have a similar folding pattern to the protein with which it is being compared (5).  Sequence determines structure. An E score is a measure of similarity between two proteins.  Comparing the amino acid sequence of proteins and assigning gaps as differences between the sequences determine this score.   3-Ketoacyl-CoA thiolase in Arabidopsis has an E score of 2e-111 when compared to 3-Ketoacyl-CoA thiolase in S. cerevisiae.  In order for a protein to be similar it must have an E score of less than 0.5.  3-Ketoacyl-CoA thiolase in Arabidopsis is similar to 3-Ketoacyl-CoA thiolase in S. cerevisiae (6).

The main structural difference between the two thiolases occurs due to a disulfide bridge that forms using the active site i.e the Cysteines that hold the substrate in place so that the thiolytic cleavage can occur.   This blocks the active site and causes a change in the loop that caps the active site and a consequent rearrangement of dimer association (8). This inactivated version of the Arabidopsis thiolase has only eleven hydrogen bonds compared to the activated dimer that has forty-one hydrogen bonds. The Arabidopsis  thiolase also contains a different ligand than the yeast thiolase.  Yeast contains (4R)-2-methylpentane- 2,4-diol that interacts near Cys-182 via a hydrogen bond to the carbonyl oxygen of the peptide backbone (9).  Arabidopsis contains a different ligand.  This ligand is 1,2-ethanediol.  It is not known where this ligand associates (10). Both of these thiolases have a ligand that contains two hydroxyl groups. These hydroxyl groups most likely hydrogen bond to a specific amino acid to cause a conformational change, activating the protein.