Applied Microbiology and Biotechnology

, Volume 97, Issue 21, pp 9439–9449 | Cite as

Comparison of pyruvate decarboxylases from Saccharomyces cerevisiae and Komagataella pastoris (Pichia pastoris)

  • Praveen Kumar Agarwal
  • Vanita Uppada
  • Santosh B. Noronha
Biotechnologically relevant enzymes and proteins

Abstract

Pyruvate decarboxylases (PDCs) are a class of enzymes which carry out the non-oxidative decarboxylation of pyruvate to acetaldehyde. These enzymes are also capable of carboligation reactions and can generate chiral intermediates of substantial pharmaceutical interest. Typically, the decarboxylation and carboligation processes are carried out using whole cell systems. However, fermentative organisms such as Saccharomyces cerevisiae are known to contain several PDC isozymes; the precise suitability and role of each of these isozymes in these processes is not well understood. S. cerevisiae has three catalytic isozymes of pyruvate decarboxylase (ScPDCs). Of these, ScPDC1 has been investigated in detail by various groups with the other two catalytic isozymes, ScPDC5 and ScPDC6 being less well characterized. Pyruvate decarboxylase activity can also be detected in the cell lysates of Komagataella pastoris, a Crabtree-negative yeast, and consequently it is of interest to investigate whether this enzyme has different kinetic properties. This is also the first report of the expression and functional characterization of pyruvate decarboxylase from K. pastoris (PpPDC). This investigation helps in understanding the roles of the three isozymes at different phases of S. cerevisiae fermentation as well as their relevance for ethanol and carboligation reactions. The kinetic and physical properties of the four isozymes were determined using similar conditions of expression and characterization. ScPDC5 has comparable decarboxylation efficiency to that of ScPDC1; however, the former has the highest rate of reaction, and thus can be used for industrial production of ethanol. ScPDC6 has the least decarboxylation efficiency of all three isozymes of S. cerevisiae. PpPDC in comparison to all isozymes of S. cerevisiae is less efficient at decarboxylation. All the enzymes exhibit allostery, indicating that they are substrate activated.

Keywords

Pyruvate decarboxylase S. cerevisiae K. pastoris Decarboxylation Carboligation Phenyl acetyl carbinol 

References

  1. Arjunan P, Umland T, Dyda F, Swaminathan S, Furey W, Sax M, Farrenkopf B, Gao Y, Zhang D, Jordan F (1996) Crystal structure of the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from the yeast Saccharomyces cerevisiae at 2.3 Å resolution. J Mol Bio 256:590–600CrossRefGoogle Scholar
  2. Baburina I, Gao YH, Hu ZX, Jordan F, Hohmann S, Furey W (1994) Substrate activation of brewers-yeast pyruvate decarboxylase is abolished by mutation of cysteine-221 to serine. Biochem 33:5630–5635CrossRefGoogle Scholar
  3. Baburina I, Moore DJ, Volkov A, Kahyaoglu A, Jordan F, Mendelsohn R (1996) Three of four cysteines, including that responsible for substrate activation, are ionized at pH 6.0 in yeast pyruvate decarboxylase: evidence from Fourier transform infrared and isoelectric focusing studies. Biochem 35:10249–10255CrossRefGoogle Scholar
  4. Baburina I, Li HJ, Bennion B, Furey W, Jordan F (1998) Interdomain information transfer during substrate activation of yeast pyruvate decarboxylase: the interaction between cysteine 221 and histidine 92. Biochem 37:1235–1244CrossRefGoogle Scholar
  5. Breunig KD, Bolotin-Fukuhara M, Bianchi MM, Bourgarel D, Falcone C, Ferrero II, Frontali L, Goffrini P, Krijger JJ, Mazzoni C, Milkowski C, Steensma HY, Wesolowski-Louvel M, Zeeman AM (2000) Regulation of primary carbon metabolism in Kluyveromyces lactis. Enzyme Microb Technol 26:771–780PubMedCrossRefGoogle Scholar
  6. Bruhn H, Pohl M, Grotzinger J, Kula MR (1995) The replacement of Trp392 by alanine influences the decarboxylase/carboligase activity and stability of pyruvate decarboxylase from Zymomonas mobilis. Eur J Biochem 234:650–655PubMedCrossRefGoogle Scholar
  7. Candy JM, Duggleby RG (1998) Structure and properties of pyruvate decarboxylase and site-directed mutagenesis of the Zymomonas mobilis enzyme. Biochim Biophys Acta Protein Struct Mol Enzymol 1385:323–338CrossRefGoogle Scholar
  8. Chang YH, Chang AK, Nixon PF, Duggleby RG (2001) Site-directed mutagenesis of the ionizable groups in the active site of Zymomonas mobilis pyruvate decarboxylase. Eur J Biochem 268:3558–3565PubMedCrossRefGoogle Scholar
  9. De Schutter K, Lin YC, Tiels P, Van HA, Glinka S, Weber-Lehmann J, Rouze P, Van de Peer Y, Callewaert N (2009) Genome sequence of the recombinant protein production host Pichia pastoris. Nat Biotechnol 27:561–566PubMedCrossRefGoogle Scholar
  10. Dobritzsch D, König S, Schneider G, Lu G (1998) High resolution crystal structure of pyruvate decarboxylase from Zymomonas mobilis. J Biol Chem 273:20196–20204PubMedCrossRefGoogle Scholar
  11. Erasmus DJ, van der Merwe GK, van Vuuren HJ (2003) Genome-wide expression analyses: metabolic adaptation of Saccharomyces cerevisiae to high sugar stress. FEMS Yeast Res 3:375–399PubMedCrossRefGoogle Scholar
  12. Fauchon M, Lagniel G, Aude JC, Lombardia L, Soularue P, Petat C, Marguerie G, Sentenac A, Werner M, Labarre J (2002) Sulfur sparing in the yeast proteome in response to sulfur demand. Mol Cell 9:713–723PubMedCrossRefGoogle Scholar
  13. Flikweert MT, van der Zanden L, Janssen WM, Steensma HY, van Dijken JP, Pronk JT (1996) Pyruvate decarboxylase: an indispensable enzyme for growth of Saccharomyces cerevisiae on glucose. Yeast 12:247–257PubMedCrossRefGoogle Scholar
  14. Fredlund E, Beerlage C, Melin P, Schnurer J, Passoth V (2006) Oxygen and carbon source-regulated expression of PDC and ADH genes in the respiratory yeast Pichia anomala. Yeast 23:1137–1149PubMedCrossRefGoogle Scholar
  15. Hildebrandt G, Klavehn W (1932) Verfahren zur Herstellung von 1-l-Phenyl-2-methylamino-1-ol. German Patent 548 459Google Scholar
  16. Hohmann S (1993) Characterisation of PDC2, a gene necessary for high level expression of pyruvate decarboxylase structural genes in Saccharomyces cerevisiae. Mol Gen Genet 241:657–666PubMedCrossRefGoogle Scholar
  17. Hohmann S, Cederberg H (1990) Autoregulation may control the expression of yeast pyruvate decarboxylase structural genes PDC1 and PDC5. Eur J Biochem 188:615–621PubMedCrossRefGoogle Scholar
  18. Hübner G, Weidhase R, Schellenberger A (1978) Mechanism of substrate activation of pyruvate decarboxylase—1st approach. Eur J Biochem 92:175–181PubMedCrossRefGoogle Scholar
  19. Iding H, Siegert P, Mesch K, Pohl M (1998) Application of α-keto acid decarboxylases in biotransformations. Biochim Biophys Acta 1385:307–322PubMedCrossRefGoogle Scholar
  20. Inan M, Meagher M (2001) The effect of ethanol and acetate on protein expression in Pichia pastoris. J Biosci Bioeng 92:337–341PubMedGoogle Scholar
  21. Ishchuk OP, Voronovsky AY, Stasyk OV, Gayda GZ, Gonchar MV, Abbas CA, Sibirny AA (2008) Overexpression of pyruvate decarboxylase in the yeast Hansenula polymorpha results in increased ethanol yield in high-temperature fermentation of xylose. FEMS Yeast Res 8:1164–1174PubMedCrossRefGoogle Scholar
  22. Ito H, Ukuda Y, Urata K, Imura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168PubMedGoogle Scholar
  23. König S (1998) Subunit structure, function and organisation of pyruvate decarboxylases from various organisms. Biochim Biophys Acta Protein Struct Mol Enzymol 1385:271–286CrossRefGoogle Scholar
  24. Kresze GB, Ronft H (1981) Pyruvate dehydrogenase complex from baker's yeast. 2. Molecular structure, dissociation, and implications for the origin of mitochondria. Eur J Biochem 119:581–587PubMedCrossRefGoogle Scholar
  25. Krieger F, Spinka M, Golbik R, Hübner G, König S (2002) Pyruvate decarboxylase from Kluyveromyces lactis. An enzyme with an extraordinary substrate activation behaviour. Eur J Biochem 269:3256–3263PubMedCrossRefGoogle Scholar
  26. Kutter S, Wille G, Relle S, Weiss MS, Hübner G, König S (2006) The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis. Implications for the substrate activation mechanism of this enzyme. FEBS J 273:4199–4209PubMedCrossRefGoogle Scholar
  27. Lohmann K, Schuster P (1937) Untersuchungen über die cocarboxylase. Biochem Z 294:188–214Google Scholar
  28. Lowe SE, Zeikus JG (1992) Purification and characterization of pyruvate decarboxylase from Sarcina ventriculi. J Gen Microbiol 138:803–807PubMedCrossRefGoogle Scholar
  29. Lu P, Davis BP, Jeffries TW (1998) Cloning and characterization of two pyruvate decarboxylase genes from Pichia stipitis CBS 6054. Appl Environ Microbiol 64:94–97PubMedGoogle Scholar
  30. Lu GG, Dobritzsch D, Baumann S, Schneider G, König S (2000) The structural basis of substrate activation in yeast pyruvate decarboxylase—a crystallographic and kinetic study. Eur J Biochem 267:861–868PubMedCrossRefGoogle Scholar
  31. Mattanovich D, Graf A, Stadlmann J, Dragosits M, Redl A, Maurer M, Kleinheinz M, Sauer M, Altmann F, Gasser B (2009) Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris. Microb Cell Fact 8:53PubMedCrossRefGoogle Scholar
  32. Mojzita D, Hohmann S (2006) PDC2 coordinates expression of the THI regulon in the yeast Saccharomyces cerevisiae. Mol Genet Genomics 276:147–161PubMedCrossRefGoogle Scholar
  33. Mumberg D, Müller R, Funk M (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156:119–122PubMedCrossRefGoogle Scholar
  34. Muller YA, Indqvist Y, Furey W, Schulz GE, Jordan F, Schneider G (1993) A thiamin diphosphate binding fold revealed by comparison of the crystal structures of transketolase, pyruvate oxidase and pyruvate decarboxylase. Structure 1:95–103PubMedCrossRefGoogle Scholar
  35. Pereira Y, Lagniel G, Godat E, Baudouin-Cornu P, Junot C, Labarre J (2008) Chromate causes sulfur starvation in yeast. Toxicol Sci 106:400–412PubMedCrossRefGoogle Scholar
  36. Reynolds JEF (1989) Martindale, the extra pharmacopoeia. Pharmaceutical Press, London, UK, pp 1462–1463Google Scholar
  37. Schaaff I, Green JB, Gozalbo D, Hohmann S (1989) A deletion of the PDC1 gene for pyruvate decarboxylase of yeast causes a different phenotype than previously isolated point mutations. Curr Genet 15:75–81PubMedCrossRefGoogle Scholar
  38. Schellenberger A (1967) Structure and mechanism of action of the active center of yeast pyruvate decarboxylase. Angew Chem Int Ed 6:1024–1035CrossRefGoogle Scholar
  39. Schmitt ME, Brown TA, Trumpower BL (1990) A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res 18:3091–3092PubMedCrossRefGoogle Scholar
  40. Singer TP, Pensky J (1951) Acetoin synthesis by highly purified a-carboxylase. Arch Biochem Biophys 31:457–459PubMedCrossRefGoogle Scholar
  41. Ullrich J, Wittorf JH, Gubler CJ (1966) Molecular weight and coenzyme content of pyruvate decarboxylase from brewer's yeast. Biochim Biophys Acta 113:595–604PubMedCrossRefGoogle Scholar
  42. van Urk H, Voll WSL, Scheffers WA, van Dijken JP (1990) Transient-state analysis of metabolic fluxes in Crabtree-positive and Crabtree-negative yeasts. Appl Environ Microbiol 56:281–287PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Praveen Kumar Agarwal
    • 1
    • 3
  • Vanita Uppada
    • 1
  • Santosh B. Noronha
    • 1
    • 2
  1. 1.Department of Biosciences and BioengineeringIIT BombayMumbaiIndia
  2. 2.Department of Chemical engineeringIIT BombayMumbaiIndia
  3. 3.Gennova BiopharmaceuticalsPuneIndia

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