Thiolases, also known asacetyl-coenzyme A acetyltransferases (ACAT), are enzymes which convert two units ofacetyl-CoA toacetoacetyl CoA in themevalonate pathway.

Thiolases are ubiquitousenzymes that have key roles in many vital biochemical pathways, including thebeta oxidation pathway of fatty acid degradation and various biosynthetic pathways.^[1] Members of the thiolase family can be divided into two broad categories: degradative thiolases (EC 2.3.1.16) and biosynthetic thiolases (EC 2.3.1.9). These two different types of thiolase are found both ineukaryotes and inprokaryotes: acetoacetyl-CoA thiolase (EC:2.3.1.9) and3-ketoacyl-CoA thiolase (EC:2.3.1.16).3-ketoacyl-CoA thiolase (also called thiolase I) has a broad chain-length specificity for its substrates and is involved in degradative pathways such as fatty acid beta-oxidation. Acetoacetyl-CoA thiolase (also called thiolase II) is specific for thethiolysis ofacetoacetyl-CoA and involved in biosynthetic pathways such asbeta-hydroxybutyric acid synthesis orsteroid biogenesis.

The formation of a carbon–carbon bond is a key step in the biosynthetic pathways by whichfatty acids andpolyketide are made. The thiolase superfamilyenzymes catalyse the carbon–carbon-bond formation via a thioester-dependentClaisen condensation^[2] reaction mechanism.^[3]

Thiolases area family of evolutionarily relatedenzymes. Two different types of thiolase^[4][5][6] are found both in eukaryotes and in prokaryotes:acetoacetyl-CoA thiolase (EC2.3.1.9) and3-ketoacyl-CoA thiolase (EC2.3.1.16). 3-ketoacyl-CoA thiolase (also called thiolase I) has a broad chain-length specificity for its substrates and is involved in degradative pathways such as fatty acid beta-oxidation. Acetoacetyl-CoA thiolase (also called thiolase II) is specific for the thiolysis ofacetoacetyl-CoA and involved in biosynthetic pathways such as poly beta-hydroxybutyrate synthesis or steroid biogenesis.

In eukaryotes, there are two forms of 3-ketoacyl-CoA thiolase: one located in the mitochondrion and the other in peroxisomes.

There are two conserved cysteine residues important for thiolase activity. The first located in the N-terminal section of the enzymes are involved in the formation of an acyl-enzyme intermediate; the second located at the C-terminal extremity is the active site base involved in deprotonation in the condensation reaction.

Mammalian nonspecific lipid-transfer protein (nsL-TP) (also known assterol carrier protein 2) is a protein which seems to exist in two different forms: a 14 Kd protein (SCP-2) and a larger 58 Kd protein (SCP-x). The former is found in the cytoplasm or the mitochondria and is involved in lipid transport; the latter is found inperoxisomes. The C-terminal part of SCP-x is identical to SCP-2 while the N-terminal portion is evolutionary related to thiolases.^[6]

Thioesters are more reactive than oxygen esters and are common intermediates in fatty-acid metabolism.^[7] These thioesters are made by conjugating the fatty acid with the free SH group of thepantetheine moiety of eithercoenzyme A (CoA) oracyl carrier protein (ACP).

All thiolases, whether they are biosynthetic or degradative in vivo, preferentially catalyze the degradation of 3-ketoacyl-CoA to form acetyl-CoA and a shortened acyl-CoA species, but are also capable of catalyzing the reverseClaisen condensation reaction (reflecting the negative Gibbs energy change of the degradation, which is independent of the thiolase catalyzing the reaction). It is well established from studies on the biosynthetic thiolase from Z. ramigera that the thiolase reaction occurs in two steps and follows ping-pong kinetics.^[8] In the first step of both the degradative and biosynthetic reactions, the nucleophilic Cys89 (or its equivalent) attacks the acyl-CoA (or 3-ketoacyl-CoA) substrate, leading to the formation of a covalent acyl-enzyme intermediate.^[9] In the second step, the addition of CoA (in the degradative reaction) or acetyl-CoA (in the biosynthetic reaction) to the acyl–enzyme intermediate triggers the release of the product from the enzyme.^[10] Each of the tetrahedral reaction intermediates that occur during transfer of an acetyl group to and from the nucleophilic cysteine, respectively, have been observed in X-ray crystal structures of biosynthetic thiolase from A. fumigatus.^[11]

Most enzymes of the thiolase superfamily aredimers. However, monomers have not been observed.Tetramers are observed only in the thiolase subfamily and, in these cases, the dimers have dimerized to become tetramers. The crystal structure of the tetrameric biosynthetic thiolase fromZoogloea ramigera has been determined at 2.0 Å resolution. The structure contains a striking and novel ‘cage-like’ tetramerization motif, which allows for some hinge motion of the two tight dimers with respect to each other. The enzyme tetramer is acetylated at Cys89 and has a CoA molecule bound in each of itsactive-site pockets.^[12]

Ineukaryotic cells, especially in mammalian cells, thiolases exhibit diversity in intracellular localization related to their metabolic functions as well as in substrate specificity. For example, they contribute to fatty-acid β-oxidation inperoxisomes andmitochondria,ketone body metabolism in mitochondria,^[13] and the early steps ofmevalonate pathway in peroxisomes andcytoplasm.^[14] In addition to biochemical investigations, analyses of genetic disorders have made clear the basis of their functions.^[15] Genetic studies have identified a three-thiolase system in the yeastCandida tropicalis, which has thiolase activity in peroxisomes, where it may participate in beta oxidation, and in the cytosol, where it participates in the mevalonate pathway.^[16][17] Thiolase is of central importance in key enzymatic pathways such as fatty-acid, steroid and polyketide synthesis. The detailed understanding of its structural biology is of great medical relevance, for example, for a better understanding of the diseases caused by genetic deficiencies of these enzymes and for the development of new antibiotics.^[18] Harnessing the complicated catalytic versatility of the polyketide synthases for the synthesis of biologically and medically relevant natural products is also an important future perspective of the studies of the enzymes of this superfamily.^[19]

Mitochondrial acetoacetyl-CoA thiolase deficiency, known earlier asβ-ketothiolase deficiency,^[20] is aninborn error of metabolism involvingisoleucine catabolism and ketone body metabolism. The major clinical manifestations of this disorder are intermittentketoacidosis but the long-term clinical consequences, apparently benign, are not well documented. Mitochondrial acetoacetyl-CoA thiolase deficiency is easily diagnosed by urinary organic acid analysis and can be confirmed by enzymatic analysis of cultured skin fibroblasts or blood leukocytes.^[21]

β-Ketothiolase Deficiency has a variable presentation. Most affected patients present between 5 and 24 months of age with symptoms of severe ketoacidosis. Symptoms can be initiated by a dietary protein load, infection or fever. Symptoms progress from vomiting to dehydration and ketoacidosis.^[22] Neutropenia and thrombocytopenia may be present, as can moderate hyperammonemia. Blood glucose is typically normal, but can be low or high in acute episodes.^[23] Developmental delay may occur, even before the first acute episode, and bilateral striatalnecrosis of thebasal ganglia has been seen on brainMRI.

• Acetyl-CoA+C-Acetyltransferase at the U.S. National Library of MedicineMedical Subject Headings (MeSH)
• Overview of all the structural information available in thePDB forUniProt:P28790 (3-ketoacyl-CoA thiolase) at thePDBe-KB.
• Overview of all the structural information available in thePDB forUniProt:Q56WD9 (3-ketoacyl-CoA thiolase 2, peroxisomal ) at thePDBe-KB.

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