Thiolase
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Thiolases, also known as acetyl-coenzyme A acetyltransferases (ACAT), are enzymes which convert two units of
acetyl-CoA Acetyl-CoA (acetyl coenzyme A) is a molecule that participates in many biochemical reactions in protein, carbohydrate and lipid metabolism. Its main function is to deliver the acetyl group to the citric acid cycle (Krebs cycle) to be oxidation, o ...
to
acetoacetyl CoA Acetoacetyl CoA is the precursor of HMG-CoA in the mevalonate pathway, which is essential for Cholesterol#Biosynthesis, cholesterol biosynthesis. It also takes a similar role in the ketone bodies synthesis (ketogenesis) pathway of the liver. In the ...
in the
mevalonate pathway The mevalonate pathway, also known as the isoprenoid pathway or HMG-CoA reductase pathway is an essential metabolic pathway present in eukaryotes, archaea, and some bacteria. The pathway produces two five-carbon building blocks called isopentenyl ...
. Thiolases are ubiquitous
enzymes An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as pro ...
that have key roles in many vital biochemical pathways, including the
beta oxidation In biochemistry and metabolism, beta oxidation (also β-oxidation) is the catabolic process by which fatty acid molecules are broken down in the cytosol in prokaryotes and in the mitochondria in eukaryotes to generate acetyl-CoA. Acetyl-CoA enter ...
pathway of fatty acid degradation and various biosynthetic pathways. 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 in
eukaryotes The eukaryotes ( ) constitute the domain of Eukaryota or Eukarya, organisms whose cells have a membrane-bound nucleus. All animals, plants, fungi, seaweeds, and many unicellular organisms are eukaryotes. They constitute a major group of ...
and in
prokaryotes A prokaryote (; less commonly spelled procaryote) is a single-celled organism whose cell lacks a nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Ancient Greek (), meaning 'before', and (), meaning 'nut' ...
: acetoacetyl-CoA thiolase (EC:2.3.1.9) and 3-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 the thiolysis of
acetoacetyl-CoA Acetoacetyl CoA is the precursor of HMG-CoA in the mevalonate pathway, which is essential for cholesterol biosynthesis. It also takes a similar role in the ketone bodies synthesis ( ketogenesis) pathway of the liver. In the ketone bodies digestion ...
and involved in biosynthetic pathways such as beta-hydroxybutyric acid synthesis or
steroid A steroid is an organic compound with four fused compound, fused rings (designated A, B, C, and D) arranged in a specific molecular configuration. Steroids have two principal biological functions: as important components of cell membranes t ...
biogenesis. The formation of a carbon–carbon bond is a key step in the biosynthetic pathways by which
fatty acids In chemistry, in particular in biochemistry, a fatty acid is a carboxylic acid with an aliphatic chain, which is either saturated or unsaturated. Most naturally occurring fatty acids have an unbranched chain of an even number of carbon atoms, ...
and
polyketide In organic chemistry, polyketides are a class of natural products derived from a Precursor (chemistry), precursor molecule consisting of a Polymer backbone, chain of alternating ketone (, or Carbonyl reduction, its reduced forms) and Methylene gro ...
are made. The thiolase superfamily
enzymes An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as pro ...
catalyse the carbon–carbon-bond formation via a thioester-dependent
Claisen condensation The Claisen condensation is a carbon–carbon bond forming reaction that occurs between two esters or one ester and another carbonyl compound in the presence of a strong base. The reaction produces a β-keto ester or a β- diketone. It is named ...
reaction mechanism.


Function

Thiolases are a family of evolutionarily related
enzymes An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as pro ...
. Two different types of thiolase are found both in eukaryotes and in prokaryotes:
acetoacetyl-CoA thiolase Thiolases, also known as acetyl-coenzyme A acetyltransferases (ACAT), are enzymes which convert two units of acetyl-CoA to acetoacetyl CoA in the mevalonate pathway. Thiolases are ubiquitous enzymes that have key roles in many vital biochemi ...
() and 3-ketoacyl-CoA thiolase (). 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 of
acetoacetyl-CoA Acetoacetyl CoA is the precursor of HMG-CoA in the mevalonate pathway, which is essential for cholesterol biosynthesis. It also takes a similar role in the ketone bodies synthesis ( ketogenesis) pathway of the liver. In the ketone bodies digestion ...
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.


Isozymes

Mammalian nonspecific lipid-transfer protein (nsL-TP) (also known as sterol 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 in
peroxisomes A peroxisome () is a membrane-bound organelle, a type of microbody, found in the cytoplasm of virtually all eukaryotic cells. Peroxisomes are oxidative organelles. Frequently, molecular oxygen serves as a co-substrate, from which hydrogen pe ...
. The C-terminal part of SCP-x is identical to SCP-2 while the N-terminal portion is evolutionary related to thiolases.


Mechanism

Thioesters are more reactive than oxygen esters and are common intermediates in fatty-acid metabolism. These thioesters are made by conjugating the fatty acid with the free SH group of the pantetheine moiety of either
coenzyme A Coenzyme A (CoA, SHCoA, CoASH) is a coenzyme, notable for its role in the Fatty acid metabolism#Synthesis, synthesis and Fatty acid metabolism#.CE.B2-Oxidation, oxidation of fatty acids, and the oxidation of pyruvic acid, pyruvate in the citric ac ...
(CoA) or acyl 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 reverse
Claisen condensation The Claisen condensation is a carbon–carbon bond forming reaction that occurs between two esters or one ester and another carbonyl compound in the presence of a strong base. The reaction produces a β-keto ester or a β- diketone. It is named ...
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. 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. 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. 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.


Structure

Most enzymes of the thiolase superfamily are dimers. However, monomers have not been observed.
Tetramers A tetramer () (''tetra-'', "four" + '' -mer'', "parts") is an oligomer formed from four monomers or subunits. The associated property is called ''tetramery''. An example from inorganic chemistry is titanium methoxide with the empirical formula ...
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 from '' Zoogloea 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 its active-site pockets.


Biological function

In
eukaryotic The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...
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 in
peroxisomes A peroxisome () is a membrane-bound organelle, a type of microbody, found in the cytoplasm of virtually all eukaryotic cells. Peroxisomes are oxidative organelles. Frequently, molecular oxygen serves as a co-substrate, from which hydrogen pe ...
and
mitochondria A mitochondrion () is an organelle found in the cells of most eukaryotes, such as animals, plants and fungi. Mitochondria have a double membrane structure and use aerobic respiration to generate adenosine triphosphate (ATP), which is us ...
,
ketone body Ketone bodies are water-soluble molecules or compounds that contain the ketone groups produced from fatty acids by the liver ( ketogenesis). Ketone bodies are readily transported into tissues outside the liver, where they are converted into acet ...
metabolism in mitochondria, and the early steps of
mevalonate pathway The mevalonate pathway, also known as the isoprenoid pathway or HMG-CoA reductase pathway is an essential metabolic pathway present in eukaryotes, archaea, and some bacteria. The pathway produces two five-carbon building blocks called isopentenyl ...
in peroxisomes and
cytoplasm The cytoplasm describes all the material within a eukaryotic or prokaryotic cell, enclosed by the cell membrane, including the organelles and excluding the nucleus in eukaryotic cells. The material inside the nucleus of a eukaryotic cell a ...
. In addition to biochemical investigations, analyses of genetic disorders have made clear the basis of their functions. Genetic studies have identified a three-thiolase system in the yeast '' Candida 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. 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. 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.


Disease relevance

Mitochondrial acetoacetyl-CoA thiolase deficiency, known earlier as β-ketothiolase deficiency, is an
inborn error of metabolism Inborn errors of metabolism form a large class of genetic diseases involving congenital disorders of enzyme activities. The majority are due to defects of single genes that code for enzymes that facilitate conversion of various substances (substrat ...
involving isoleucine catabolism and ketone body metabolism. The major clinical manifestations of this disorder are intermittent
ketoacidosis Ketoacidosis is a metabolic state caused by uncontrolled production of ketone bodies that cause a metabolic acidosis. While ketosis refers to any elevation of blood ketones, ketoacidosis is a specific pathologic condition that results in changes i ...
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. 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. Neutropenia and thrombocytopenia may be present, as can moderate hyperammonemia. Blood glucose is typically normal, but can be low or high in acute episodes. Developmental delay may occur, even before the first acute episode, and bilateral striatal
necrosis Necrosis () is a form of cell injury which results in the premature death of cells in living tissue by autolysis. The term "necrosis" came about in the mid-19th century and is commonly attributed to German pathologist Rudolf Virchow, who i ...
of the
basal ganglia The basal ganglia (BG) or basal nuclei are a group of subcortical Nucleus (neuroanatomy), nuclei found in the brains of vertebrates. In humans and other primates, differences exist, primarily in the division of the globus pallidus into externa ...
has been seen on brain
MRI Magnetic resonance imaging (MRI) is a medical imaging technique used in radiology to generate pictures of the anatomy and the physiological processes inside the body. MRI scanners use strong magnetic fields, magnetic field gradients, and rad ...
.


References


External links

* * * {{InterPro content, IPR002155 Protein domains