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A biosynthetic thiolase in complex with a reaction intermediate: the crystal structure provides new insights into the catalytic mechanism.

Modis Y, Wierenga RK
Structure 7 1279-90 (1999)
Cited: 32 times
EuropePMC logo PMID: 10545327

Abstract

Background

Thiolases are ubiquitous and form a large family of dimeric or tetrameric enzymes with a conserved, five-layered alphabetaalphabetaalpha catalytic domain. Thiolases can function either degradatively, in the beta-oxidation pathway of fatty acids, or biosynthetically. Biosynthetic thiolases catalyze the biological Claisen condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA. This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in a wide range of biosynthetic pathways, including those that generate cholesterol, steroid hormones, and various energy-storage molecules.

Results

The crystal structure of the tetrameric biosynthetic thiolase from Zoogloea ramigera has been determined at 2.0 A 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 protein crystals were flash-frozen after a short soak with the enzyme's substrate, acetoacetyl-CoA. A reaction intermediate was thus trapped: the enzyme tetramer is acetylated at Cys89 and has a CoA molecule bound in each of its active-site pockets.

Conclusion

The shape of the substrate-binding pocket reveals the basis for the short-chain substrate specificity of the enzyme. The active-site architecture, and in particular the position of the covalently attached acetyl group, allow a more detailed reaction mechanism to be proposed in which Cys378 is involved in both steps of the reaction. The structure also suggests an important role for the thioester oxygen atom of the acetylated enzyme in catalysis.

Articles - 1qfl mentioned but not cited (2)

  1. Structural basis for channelling mechanism of a fatty acid beta-oxidation multienzyme complex. Ishikawa M, Tsuchiya D, Oyama T, Tsunaka Y, Morikawa K. EMBO J 23 2745-2754 (2004)
  2. Coenzyme A-free activity, crystal structure, and rational engineering of a promiscuous β-ketoacyl thiolase from Ralstonia eutropha. Fage CD, Meinke JL, Keatinge-Clay AT. J Mol Catal B Enzym 121 113-121 (2015)


Reviews citing this publication (4)

  1. The thiolase superfamily: condensing enzymes with diverse reaction specificities. Haapalainen AM, Meriläinen G, Wierenga RK. Trends Biochem Sci 31 64-71 (2006)
  2. beta-oxidation - strategies for the metabolism of a wide variety of acyl-CoA esters. Hiltunen JK, Qin Y. Biochim Biophys Acta 1484 117-128 (2000)
  3. Thiol switches in mitochondria: operation and physiological relevance. Riemer J, Schwarzländer M, Conrad M, Herrmann JM. Biol Chem 396 465-482 (2015)
  4. Reversal of β-oxidative pathways for the microbial production of chemicals and polymer building blocks. Kallscheuer N, Polen T, Bott M, Marienhagen J. Metab Eng 42 33-42 (2017)

Articles citing this publication (26)

  1. Structures of beta-ketoacyl-acyl carrier protein synthase I complexed with fatty acids elucidate its catalytic machinery. Olsen JG, Kadziola A, von Wettstein-Knowles P, Siggaard-Andersen M, Larsen S. Structure 9 233-243 (2001)
  2. Acetoacetyl-CoA thiolase regulates the mevalonate pathway during abiotic stress adaptation. Soto G, Stritzler M, Lisi C, Alleva K, Pagano ME, Ardila F, Mozzicafreddo M, Cuccioloni M, Angeletti M, Ayub ND. J Exp Bot 62 5699-5711 (2011)
  3. Identification and analysis of the polyhydroxyalkanoate-specific beta-ketothiolase and acetoacetyl coenzyme A reductase genes in the cyanobacterium Synechocystis sp. strain PCC6803. Taroncher-Oldenburg G, Nishina K, Stephanopoulos G. Appl Environ Microbiol 66 4440-4448 (2000)
  4. High resolution crystal structures of human cytosolic thiolase (CT): a comparison of the active sites of human CT, bacterial thiolase, and bacterial KAS I. Kursula P, Sikkilä H, Fukao T, Kondo N, Wierenga RK. J Mol Biol 347 189-201 (2005)
  5. FadA5 a thiolase from Mycobacterium tuberculosis: a steroid-binding pocket reveals the potential for drug development against tuberculosis. Schaefer CM, Lu R, Nesbitt NM, Schiebel J, Sampson NS, Kisker C. Structure 23 21-33 (2015)
  6. Thiolase engineering for enhanced butanol production in Clostridium acetobutylicum. Mann MS, Lütke-Eversloh T. Biotechnol Bioeng 110 887-897 (2013)
  7. Characterization of six mutations in five Spanish patients with mitochondrial acetoacetyl-CoA thiolase deficiency: effects of amino acid substitutions on tertiary structure. Fukao T, Nakamura H, Nakamura K, Perez-Cerda C, Baldellou A, Barrionuevo CR, Castello FG, Kohno Y, Ugarte M, Kondo N. Mol Genet Metab 75 235-243 (2002)
  8. Redox-switch regulatory mechanism of thiolase from Clostridium acetobutylicum. Kim S, Jang YS, Ha SC, Ahn JW, Kim EJ, Lim JH, Cho C, Ryu YS, Lee SK, Lee SY, Kim KJ. Nat Commun 6 8410 (2015)
  9. A novel single-base substitution (380C>T) that activates a 5-base downstream cryptic splice-acceptor site within exon 5 in almost all transcripts in the human mitochondrial acetoacetyl-CoA thiolase gene. Nakamura K, Fukao T, Perez-Cerda C, Luque C, Song XQ, Naiki Y, Kohno Y, Ugarte M, Kondo N. Mol Genet Metab 72 115-121 (2001)
  10. Identification, purification and characterization of an acetoacetyl-CoA thiolase from rat liver peroxisomes. Antonenkov VD, Croes K, Waelkens E, Van Veldhoven PP, Mannaerts GP. Eur J Biochem 267 2981-2990 (2000)
  11. Archaeal acetoacetyl-CoA thiolase/HMG-CoA synthase complex channels the intermediate via a fused CoA-binding site. Vögeli B, Engilberge S, Girard E, Riobé F, Maury O, Erb TJ, Shima S, Wagner T. Proc Natl Acad Sci U S A 115 3380-3385 (2018)
  12. Haloarchaeal-type β-ketothiolases involved in Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in Haloferax mediterranei. Hou J, Feng B, Han J, Liu H, Zhao D, Zhou J, Xiang H. Appl Environ Microbiol 79 5104-5111 (2013)
  13. The crystal structure of human mitochondrial 3-ketoacyl-CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities. Kiema TR, Harijan RK, Strozyk M, Fukao T, Alexson SE, Wierenga RK. Acta Crystallogr D Biol Crystallogr 70 3212-3225 (2014)
  14. Molecular and functional characterization of an acetyl-CoA acetyltransferase from the adzuki bean borer moth Ostrinia scapulalis (Lepidoptera: Crambidae). Fujii T, Ito K, Katsuma S, Nakano R, Shimada T, Ishikawa Y. Insect Biochem Mol Biol 40 74-78 (2010)
  15. Crystal structure and biochemical characterization of beta-keto thiolase B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16. Kim EJ, Son HF, Kim S, Ahn JW, Kim KJ. Biochem Biophys Res Commun 444 365-369 (2014)
  16. The sulfur atoms of the substrate CoA and the catalytic cysteine are required for a productive mode of substrate binding in bacterial biosynthetic thiolase, a thioester-dependent enzyme. Meriläinen G, Schmitz W, Wierenga RK, Kursula P. FEBS J 275 6136-6148 (2008)
  17. Ligand-induced domain rearrangement of fatty acid beta-oxidation multienzyme complex. Tsuchiya D, Shimizu N, Ishikawa M, Suzuki Y, Morikawa K. Structure 14 237-246 (2006)
  18. Improved production of adipate with Escherichia coli by reversal of β-oxidation. Kallscheuer N, Gätgens J, Lübcke M, Pietruszka J, Bott M, Polen T. Appl Microbiol Biotechnol 101 2371-2382 (2017)
  19. Rational design of thiolase substrate specificity for metabolic engineering applications. Bonk BM, Tarasova Y, Hicks MA, Tidor B, Prather KLJ. Biotechnol Bioeng 115 2167-2182 (2018)
  20. Crystal structure of a monomeric thiolase-like protein type 1 (TLP1) from Mycobacterium smegmatis. Janardan N, Harijan RK, Wierenga RK, Murthy MR. PLoS One 7 e41894 (2012)
  21. OleA Glu117 is key to condensation of two fatty-acyl coenzyme A substrates in long-chain olefin biosynthesis. Jensen MR, Goblirsch BR, Christenson JK, Esler MA, Mohamed FA, Wackett LP, Wilmot CM. Biochem J 474 3871-3886 (2017)
  22. Crystal structure and biochemical characterization of a 3-ketoacyl-CoA thiolase from Ralstoniaeutropha H16. Kim J, Kim KJ. Int J Biol Macromol 82 425-431 (2016)
  23. Crystal structure and biochemical properties of ReH16_A1887, the 3-ketoacyl-CoA thiolase from Ralstonia eutropha H16. Kim J, Kim KJ. Biochem Biophys Res Commun 459 547-552 (2015)
  24. Effects of toxic Microcystis aeruginosa on the silver carp Hypophthalmichtys molitrix revealed by hepatic RNA-seq and miRNA-seq. Hu M, Qu X, Pan L, Fu C, Jia P, Liu Q, Wang Y. Sci Rep 7 10456 (2017)
  25. Integrated omics approach to unveil antifungal bacterial polyynes as acetyl-CoA acetyltransferase inhibitors. Lin CC, Hoo SY, Ma LT, Lin C, Huang KF, Ho YN, Sun CH, Lee HJ, Chen PY, Shu LJ, Wang BW, Hsu WC, Ko TP, Yang YL. Commun Biol 5 454 (2022)
  26. Structural characterization of a mitochondrial 3-ketoacyl-CoA (T1)-like thiolase from Mycobacterium smegmatis. Janardan N, Harijan RK, Kiema TR, Wierenga RK, Murthy MR. Acta Crystallogr D Biol Crystallogr 71 2479-2493 (2015)


Related citations provided by authors (1)

  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. Thompson S, Mayer F, Peoples OP, Masamune S, Sinskey A, Walsh CT Biochemistry 28 5735-5742 (1989)

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