Biology:Thiolase

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Short description: Enzymes
Thiolase, N-terminal domain
Identifiers
SymbolThiolase_N
PfamPF00108
InterProIPR002155
PROSITEPDOC00092
SCOP21pxt / SCOPe / SUPFAM
CDDcd00751
Thiolase, C-terminal domain
Identifiers
SymbolThiolase_C
PfamPF02803
InterProIPR002155
PROSITEPDOC00092
SCOP21pxt / SCOPe / SUPFAM
Mevalonate pathway

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 biochemical pathways, including the beta 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 in eukaryotes and in prokaryotes: 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 and involved in biosynthetic pathways such as beta-hydroxybutyric acid synthesis or steroid biogenesis.

The formation of a carbon–carbon bond is a key step in the biosynthetic pathways by which fatty acids and polyketide are made. The thiolase superfamily enzymes catalyse the carbon–carbon-bond formation via a thioester-dependent Claisen condensation[2] reaction mechanism.[3]

Function

Thiolases are a family of evolutionarily related enzymes. Two different types of thiolase[4][5][6] are found both in eukaryotes and in prokaryotes: 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 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

EC number Name Alternate name Isozymes Subcellular distribution
EC 2.3.1.9 Acetyl-CoA C-acetyltransferase thiolase II;
Acetoacetyl-CoA thiolase		 ACAT1 mitochondrial
ACAT2 cytosolic
EC 2.3.1.16 Acetyl-CoA C-acyltransferase thiolase I;
3-Ketoacyl-CoA thiolase;
β-Ketothiolase
3-KAT		 ACAA1 peroxisomal
ACAA2 mitochondrial
HADHB mitochondrial
EC 2.3.1.154 Propionyl-CoA C2-trimethyltridecanoyltransferase 3-Oxopristanoyl-CoA thiolase
EC 2.3.1.174 3-Oxoadipyl-CoA thiolase β-Ketoadipyl-CoA thiolase
EC 2.3.1.176 Propanoyl-CoA C-acyltransferase Peroxisomal thiolase 2 SCP2 peroxisomal/cytosolic

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. The C-terminal part of SCP-x is identical to SCP-2 while the N-terminal portion is evolutionary related to thiolases.[6]

Mechanism

Reaction catalyzed by thiolase

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 the pantetheine moiety of either coenzyme A (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 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]

Thiolase Mechanism. The two-step, ping-pong mechanism for the thiolase reaction. Red arrows indicate the biosynthetic reaction; Black arrows trace the degradative reaction. In both directions, the reaction is initiated by the nucleophilic attack of Cys89 on the substrate to form a covalent acetyl–enzyme intermediate. Cys89 is activated for nucleophilic attack by His348, which abstracts the sulfide proton of Cys89. In the second step of both the biosynthetic and degradative reactions, the substrate nucleophilically attacks the acetyl–enzyme intermediate to yield the final product and free enzyme. This nucleophilic attack is activated by Cys378, which abstracts a proton from the substrate.

Structure

Most enzymes of the thiolase superfamily are dimers. 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 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.[12]

Biological function

In eukaryotic 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 and mitochondria, ketone body metabolism in mitochondria,[13] and the early steps of mevalonate pathway in peroxisomes and cytoplasm.[14] In addition to biochemical investigations, analyses of genetic disorders have made clear the basis of their functions.[15] Genetic studies have also started to disclose the physiological functions of thiolases in the yeast Saccharomyces cerevisiae.[16] 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.[17] 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.[18]

Disease relevance

Mitochondrial acetoacetyl-CoA thiolase deficiency, known earlier as β-ketothiolase deficiency,[19] is an inborn error of metabolism involving isoleucine catabolism and ketone body metabolism. The major clinical manifestations of this disorder are intermittent ketoacidosis 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.[20]

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.[21] Neutropenia and thrombocytopenia may be present, as can moderate hyperammonemia. Blood glucose is typically normal, but can be low or high in acute episodes.[22] Developmental delay may occur, even before the first acute episode, and bilateral striatal necrosis of the basal ganglia has been seen on brain MRI.

References

  1. ↑ "Mechanistic studies on beta-ketoacyl thiolase from Zoogloea ramigera: identification of the active-site nucleophile as Cys89, its mutation to Ser89, and kinetic and thermodynamic characterization of wild-type and mutant enzymes". Biochemistry 28 (14): 5735–42. July 1989. doi:10.1021/bi00440a006. PMID 2775734. 
  2. ↑ "The Claisen condensation in biology". Nat Prod Rep 19 (5): 581–96. October 2002. doi:10.1039/b110221b. PMID 12430724. 
  3. ↑ "The thiolase superfamily: condensing enzymes with diverse reaction specificities". Trends Biochem. Sci. 31 (1): 64–71. January 2006. doi:10.1016/j.tibs.2005.11.011. PMID 16356722. 
  4. ↑ "Similarity between the amino-terminal portion of mammalian 58-kD sterol carrier protein (SCPx) and Escherichia coli acetyl-CoA acyltransferase: evidence for a gene fusion in SCPx". DNA Cell Biol. 10 (9): 695–8. November 1991. doi:10.1089/dna.1991.10.695. PMID 1755959. 
  5. ↑ "Nucleotide sequence of the fadA gene. Primary structure of 3-ketoacyl-coenzyme A thiolase from Escherichia coli and the structural organization of the fadAB operon". J. Biol. Chem. 265 (18): 10424–9. June 1990. PMID 2191949. 
  6. ↑ 6.0 6.1 "Phylogenetic analysis of the thiolase family. Implications for the evolutionary origin of peroxisomes". J. Mol. Evol. 35 (2): 147–55. August 1992. doi:10.1007/BF00183226. PMID 1354266. 
  7. ↑ Enzymatic reaction mechanisms. San Francisco: W. H. Freeman. 1979. ISBN 978-0-7167-0070-8. 
  8. ↑ Masamune, Satoru; Walsh, Christopher T.; Gamboni, Remo; Thompson, Stuart; Davis, Jeffrey T.; Williams, Simon F.; Peoples, Oliver P.; Sinskey, Anthony J. et al. (1989). "Bio-Claisen condensation catalyzed by thiolase from Zoogloea ramigera. Active site cysteine residues". J. Am. Chem. Soc. 111 (5): 1879, 1991. doi:10.1021/ja00187a053. 
  9. ↑ "The relation of acyl transfer to the overall reaction of thiolase I from porcine heart". J. Biol. Chem. 256 (14): 7371–7. July 1981. PMID 6114098. 
  10. ↑ "The 1.8 A crystal structure of the dimeric peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae: implications for substrate binding and reaction mechanism". J. Mol. Biol. 273 (3): 714–28. October 1997. doi:10.1006/jmbi.1997.1331. PMID 9402066. 
  11. ↑ Marshall, Andrew C.; Bond, Charles S.; Bruning, John B. (January 25, 2018). "Structure of Aspergillus fumigatus Cytosolic Thiolase: Trapped Tetrahedral Reaction Intermediates and Activation by Monovalent Cations.". ACS Catalysis 8 (3): 1973–1989. doi:10.1021/acscatal.7b02873. 
  12. ↑ "A biosynthetic thiolase in complex with a reaction intermediate: the crystal structure provides new insights into the catalytic mechanism". Structure 7 (10): 1279–90. October 1999. doi:10.1016/S0969-2126(00)80061-1. PMID 10545327. 
  13. ↑ Middleton B (April 1973). "The oxoacyl-coenzyme A thiolases of animal tissues". Biochem. J. 132 (4): 717–30. doi:10.1042/bj1320717. PMID 4721607. 
  14. ↑ "Metabolism of acetyl-CoA by isolated peroxisomal fractions: formation of acetate and acetoacetyl-CoA". J. Lipid Res. 32 (6): 993–9. June 1991. PMID 1682408. http://www.jlr.org/cgi/pmidlookup?view=long&pmid=1682408. 
  15. ↑ "The synthesis and characterisation of 2-methylacetoacetyl coenzyme A and its use in the identification of the site of the defect in 2-methylacetoacetic and 2-methyl-3-hydroxybutyric aciduria". Clin. Chim. Acta 128 (2–3): 291–305. March 1983. doi:10.1016/0009-8981(83)90329-7. PMID 6133656. 
  16. ↑ "Genetic evaluation of physiological functions of thiolase isoenzymes in the n-alkalane-assimilating yeast Candida tropicalis". J. Bacteriol. 180 (3): 690–8. February 1998. doi:10.1128/JB.180.3.690-698.1998. PMID 9457876. 
  17. ↑ "Inhibition of beta-ketoacyl-acyl carrier protein synthases by thiolactomycin and cerulenin. Structure and mechanism". J. Biol. Chem. 276 (9): 6551–9. March 2001. doi:10.1074/jbc.M007101200. PMID 11050088. 
  18. ↑ "An antibiotic factory caught in action". Nat. Struct. Mol. Biol. 11 (9): 888–93. September 2004. doi:10.1038/nsmb808. PMID 15286722. 
  19. ↑ "A "new" disorder of isoleucine catabolism". Lancet 2 (7737): 1289–90. December 1971. doi:10.1016/S0140-6736(71)90605-2. PMID 4143539. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=50299. 
  20. ↑ "Inborn errors of ketone body metabolism". The metabolic & molecular bases of inherited disease. New York: McGraw-Hill. 2001. pp. 2326–2356. ISBN 978-0-07-913035-8. 
  21. ↑ "Beta-ketothiolase deficiency as a cause of the "ketotic hyperglycinemia syndrome"". Pediatrics 53 (2): 221–5. February 1974. PMID 4812006. 
  22. ↑ "Acetoacetyl CoA thiolase deficiency: a cause of severe ketoacidosis in infancy simulating salicylism". J. Pediatr. 95 (2): 228–33. August 1979. doi:10.1016/S0022-3476(79)80658-7. PMID 36452. 

External links


This article incorporates text from the public domain Pfam and InterPro: IPR002155



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