Molecular and Cellular Biochemistry

, Volume 269, Issue 1, pp 27–36 | Cite as

A thermostable #x003B2;-ketothiolase of polyhydroxyalkanoates (PHAs) in Thermus thermophilus: Purification and biochemical properties

  • Anastasia A. Pantazaki
  • Andrea K. Ioannou
  • Dimitrios A. Kyriakidis


Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesised by numerous bacteria as intracellular carbon and energy storage compounds which accumulate as granules in the cytoplasm of the cells. The biosynthesis of PHAs, in the thermophilic bacterium T. thermophilus grown in a mineral medium supplemented with sodium gluconate as sole carbon source has been recently reported. Here, we report the purification at apparent homogeneity of a #x003B2;-ketoacyl-CoA thiolase from T. thermophilus, the first enzyme of the most common biosynthetic pathway for PHAs. B-Ketoacyl-CoA thiolase appeared as a single band of 45.5-kDa molecular mass on SDS/PAGE. The enzyme was purified 390-fold with 7% recovery. The native enzyme is a multimeric protein of a molecular mass of approximately of 182 kDa consisting of four identical subunits of 45.5 kDa, as identified by an in situ renaturation experiment on SDS-PAGE. The enzyme exhibited an optimal pH of approximately 8.0 and highest activity at 65 °C for both direction of the reaction. The thiolysis reaction showed a substrate inhibition at high concentrations; when one of the substrates (acetoacetyl CoA or CoA) is varied, while the concentrations of the second substrates (CoA or acetoacetyl CoA respectively) remain constant. The initial velocity kinetics showed a pattern of a family of parallel lines, which is in accordance with a ping-pong mechanism. #x003B2;-Ketothiolase had a relative low Km of 0.25 mM for acetyl-CoA and 11 μM and 25 μM for CoA and acetoacetyl-CoA, respectively. The enzyme was inhibited by treatment with 1 mM N-ethylmaleimide either in the presence or in the absence of 0.5 mM of acetyl-CoA suggesting that possibly a cysteine is located at/or near the active site of #x003B2;-ketothiolase. (Mol Cell Biochem 269: 27–36, 2005)


poly-hydroxyalkanoates (PHAs) Thermus thermophilus #x003B2;-ketothiolase purification properties 









copolymer poly(3-hydroxybutyrate-3-hydroxyvalerate);




Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Woese CR: Interpreting the universal phylogenetic tree. Proc Natl Acad Sci USA 97: 8392–8396, 2000CrossRefPubMedGoogle Scholar
  2. 2.
    Pantazaki AA, Pritsa AA, Kyriakidis DA: Biotechnologically relevant enzymes from Thermus thermophilus. Appl Microbiol Biotechnol 58: 1–12, 2002CrossRefGoogle Scholar
  3. 3.
    Hiltunen JK, Qin YM: Beta-oxidation-strategies for the metabolism of a wide veriety of acyl-CoA esters. Biochim Biophys Acta 1484: 117–128, 2000Google Scholar
  4. 4.
    Feigenbaum J, Schulz H: Thiolases of Escherichia coli: Purification and chain length specificities. J Bacteriol 122: 407–411, 1975Google Scholar
  5. 5.
    Slater S, Houmiel KL, Tran M, Mitsky TA, Taylor NB, Padgette SR, Gruys KJ: Multiple β-ketothiolases mediate polyβ-hydroxyalkanoate copolymer synthesis in Ralstonia eutropha. J Bacteriol 180: 1979–1987, 1998Google Scholar
  6. 6.
    Antonenkov VD, Van Veldhoven PP, Waelkens E, Mannaerts GP: Substrate specificities of 3-oxoacyl-CoA thiolase A and sterol carrier protein 2/3-oxoacyl-CoA thiolase purified from normal rat liver peroxisomes. J Biol Chem 272: 26023–26031, 1997CrossRefGoogle Scholar
  7. 7.
    Thompson S, Mayerl F, Peoples OP, Masamune S, Siskey AJ, Walsh CT: Mechanistic studies on β-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: 5735–5742, 1989Google Scholar
  8. 8.
    Palmer MAJ, Differding E, Gamboni R, Williams SF, Peoples OP, Walsh CT, Sinskey AJ, Masamune S: Biosynthetic thiolase from Zoogloea ramigera. Evidence for a mechanism involving Cys378 as the active site base. J Biol Chem 266: 8369–8375, 1991Google Scholar
  9. 9.
    Masamune S, Palmer MAJ, Gamboni R, Thompson S, Davis JT, Williams SF, Peoples OP, Sinskey AJ, Walsh CT: Bio-Claisen condensation catalyzed by thiolase from Zoogloea ramigera active site cysteine residues. J Am Chem Soc 111: 1878–1881, 1989Google Scholar
  10. 10.
    Berndt H, Schlegel HG: Kinetics and properties of α-ketothiolase from Chlostridium pasteurianum. Arch Microbiol 103: 21–30, 1975Google Scholar
  11. 11.
    Oeding V, Schlegel HG: β-ketothiolase from Hydrogenomonas eutropha HI6 and its significance in the regulation of poly-β-hydroxybutyrate metabolism. Biochem J 134:239–248, 1973Google Scholar
  12. 12.
    Nishimura T, Saito T, Tomita K: Purification and properties of β-ketothiolases from Zoogloea ramigera. Arch Microbiol 116: 21–27, 1978Google Scholar
  13. 13.
    Suzuki F, Zahler WL, Emerich DW: Acetoacetyl-CoA thiolase of Bradyrhizobium japonicum bacteroids: Purification and properties. Arch Biochem Biophys 254: 272–281, 1987Google Scholar
  14. 14.
    Haywood GW, Anderson AJ, Chu L, Dawes EA: Characterization of two 3-ketothiolases possessing differing substrate specificities in the polyhydroxyalkanoate synthesizing organism Alcaligenes eutrophus. FEMS Microbiol Lett 52: 91–96, 1988Google Scholar
  15. 15.
    Antonenkov VD, Croes K, Waelkens E, Van Veldhoven PP, Mannaerts GP: Identification, purification and characterization of an acetoacetyl-coA thiolase from rat liver peroxisomes. Eur J Biochem 267: 2981–2990, 2000Google Scholar
  16. 16.
    Antonenkov VD, Van Veldhoven PP, Waelkens E, Mannaerts GP: Isolation and subunit composition of native sterol carrier protein 2/3-oxoacyl-CoA thiolase from normal rat liver peroxisomes. Protein Expr Purif 18: 249–256, 2000Google Scholar
  17. 17.
    Liu T, Liu S-J, Xue Y, Ma Y: Purification and characterization of an extremely halophilic acetoacetyl-CoA thiolase from a newly isolated Halobacterium strain ZP-6. Extremophiles 6: 97–102, 2002Google Scholar
  18. 18.
    Pantazaki AA, Tambaka MG, Langlois V, Guerin P, Kyriakidis DA: Polyhydroxyalkanoate PHA biosynthesis in Thermus thermophilus: Purification and biochemical properties of PHA synthase. Mol Cell Biochem 254: 173–183, 2003Google Scholar
  19. 19.
    Oshima T, Imahori K: Description of Thermus thermophilus, com. nov., a non sporulating thermophilic bacterium from a Japanese thermal spring. Int J Syst Bacterio l24: 102–112, 1974Google Scholar
  20. 20.
    Davis JT, Moore RN, Imperiali B, Pratt AJ, Kobayashi K, Masamune S, Sinskey AJ, Walsh CT, Fukui T, Tomita K: Biosynthetic thiolase from Zoogloea ramigera. I. Preliminary characterization and analysis of proton transfer reaction. J Biol Chem 262: 82–89, 1987Google Scholar
  21. 21.
    Middleton B: The kinetic mechanism and properties of the cytoplasmic acetoacetyl-coenzyme a thiolase from rat liver. Biochem J 139: 109–121, 1973Google Scholar
  22. 22.
    Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976CrossRefPubMedGoogle Scholar
  23. 23.
    Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277: 680–685, 1970Google Scholar
  24. 24.
    Kameshita I, Fujisawa H: A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate-polyacrylamide gel. Anal Biochem 183: 139–143, 1989Google Scholar
  25. 25.
    Staack H, Binstock JF, Schulz H: Purification and properties of a pig heart thiolase with broad chain length specificity and comparison of thiolases from pig heart and E. coli. J Biol Chem 253: 1827–1831, 1978Google Scholar
  26. 26.
    Kurihara T, Ueda M, Tanaka A: Peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase from an n-alkane-utilizing yeast, Candida tropicalis: Purification and characterization. J Biochem 106: 474–478, 1989Google Scholar
  27. 27.
    Erdmann R, Kunau WH: Purification and immunolocalization of the peroxisomal 3-oxoacyl-CoA thiolase from Saccharomyces cerevisae. Yeast 10: 1173–1182, 1994Google Scholar
  28. 28.
    Kunau WH, Dommes V, Schulz H: β-oxidation of fatty acid in mitochondria, peroxisomes, and bacteria: A century of continued progress. Prog Lipid Res 34: 267–342, 1995Google Scholar
  29. 29.
    Mathieu M, Modis Y, Zeelen JP, Engel CK, Abagyan RA, Ahlberg A, Rasmussen B, Lamzin VS, Kunau W-H, Wierenga RK: The 1.8 Å crystal structure of the dimeric peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae: Implications for substrate binding and reaction mechanism. J Mol Biol 273: 714–728, 1997Google Scholar
  30. 30.
    Modis Y, Wierenga RK: Crystallographic analysis of the reaction pathway of Zoogloea ramigera biosynthetic thiolase. J Mol Biol 297: 1171–1182, 2000Google Scholar
  31. 31.
    Senior PJ, Dawes EA: The regulation of poly-β-hydroxybutyrate metabolism in Azotobacter beijerinckii. Biochem J 134: 225–238, 1973Google Scholar
  32. 32.
    Williams DR, Anderson AJ, Dawes EA: Biosynthesis of polyhydroxy-alcanoates in Rhodococcus rubber. In: H.G. Schlegel, A. Steinbuchel eds. Proceedings of the International Symposium on Bacterial PHA, Goltze-Druck, Gottingen, 1993, pp. 387–388Google Scholar
  33. 33.
    Modis Y, Wierenga RK: A biosynthetic thiolase in complex with a reaction intermediate: The crystal structure provides new insights into the catalytic mechanism. Struct Fold Des 7: 1279–1290, 1999Google Scholar
  34. 34.
    Migazuwa S, Furuta S, Osumi T, Hashimoto T, Ui N: Properties of peroxisomal 3-ketoacyl-CoA thiolase from rat liver. J Biochem 90: 511–519, 1981Google Scholar
  35. 35.
    Huth W, Jonas R, Wunderlich I, Seubert W: On the mechanism of ketogenesis and its control. Purification, kinetic mechanism and regulation of different forms of mitochondrial acetoacetyl-CoA thiolases from ox liver. Eur J Biochem 59: 475–489, 1975Google Scholar
  36. 36.
    Mothes G, Rivera IS, Babel W: Competition between P-ketothiolase and citrate synthase during polyβ-hydroxybutyrate synthesis in Methylobacterium rhodesianum. Arch Microbiol 166: 405–410, 1997Google Scholar
  37. 37.
    Gilbert HF, Lennox BJ, Mossman, CD, Carle WC: The relation of acyl transfer to the overall reaction of thiolase I from porcine heart. J Biol Chem 256: 7371–7377, 1981Google Scholar
  38. 38.
    Henne A, Bruggemann H, Raasch C, Wiezer A, Hartsch T, Liesegang H, Johann A, Lienard T, Gohl O, Martinez-Arias R, Jacobi C, Starkuviene V, Schlenczeck S, Dencker S, Huber R, Klenk HP, Kramer W, Merkl R, Gottschalk G, Fritz HJ: The genome sequence of the extreme thermophile Thermus thermophilus. Nat Biotechnol 22: 547–553, 2004Google Scholar
  39. 39.
    Aragon JJ, Lowestein JM: A survey of enzymes, which generate or use acetoacetyl thioesters in rat liver. J Boil Chem 258: 4725–4733, 1983Google Scholar

Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • Anastasia A. Pantazaki
    • 1
  • Andrea K. Ioannou
    • 1
  • Dimitrios A. Kyriakidis
    • 1
  1. 1.Laboratory of Biochemistry, Department of ChemistryAristotle University of ThessalonikiThessalonikiGreece

Personalised recommendations