Publications

• Substrate specificity of thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases in Saccharomyces cerevisiae.

  Romagnoli G., Luttik M.A., Koetter P., Pronk J.T., Daran J.M.

  Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share seq ... >> More

  Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases (2ODCs). PDC1, PDC5, and PDC6 encode differentially regulated pyruvate decarboxylase isoenzymes; ARO10 encodes a 2-oxo-acid decarboxylase with broad substrate specificity, and THI3 has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production in S. cerevisiae, the substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in a pdc1Δ pdc5Δ pdc6Δ aro10Δ thi3Δ strain, only Pdc1, Pdc5, and Pdc6 catalyzed the decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxo-butanoate, and 2-oxo-pentanoate in cell extracts. The presence of a Pdc isoenzyme was also required for the production of n-propanol and n-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production of n-propanol and n-butanol by S. cerevisiae. No decarboxylation activity was found for Thi3 with any of the substrates tested. Only Aro10 and Pdc5 catalyzed the decarboxylation of the aromatic substrate phenylpyruvate, with Aro10 showing superior kinetic properties. Aro10, Pdc1, Pdc5, and Pdc6 exhibited activity with all branched-chain and sulfur-containing 2-oxo acids tested but with markedly different decarboxylation kinetics. The high affinity of Aro10 identified it as a key contributor to the production of branched-chain and sulfur-containing fusel alcohols. << Less

  Appl. Environ. Microbiol. 78:7538-7548(2012) [PubMed] [EuropePMC]

• An investigation of the metabolism of valine to isobutyl alcohol in Saccharomyces cerevisiae.

  Dickinson J.R., Harrison S.J., Hewlins M.J.

  The metabolism of valine to isobutyl alcohol in yeast was examined by 13C nuclear magnetic resonance spectroscopy and combined gas chromatography-mass spectrometry. The product of valine transamination, alpha-ketoisovalerate, had four potential routes to isobutyl alcohol. The first, via branched-c ... >> More

  The metabolism of valine to isobutyl alcohol in yeast was examined by 13C nuclear magnetic resonance spectroscopy and combined gas chromatography-mass spectrometry. The product of valine transamination, alpha-ketoisovalerate, had four potential routes to isobutyl alcohol. The first, via branched-chain alpha-ketoacid dehydrogenase to isobutyryl-CoA is not required for the synthesis of isobutyl alcohol because abolition of branched-chain alpha-ketoacid dehydrogenase activity in an lpd1 disruption mutant did not prevent the formation of isobutyl alcohol. The second route, via pyruvate decarboxylase, is the one that is used because elimination of pyruvate decarboxylase activity in a pdc1 pdc5 pdc6 triple mutant virtually abolished isobutyl alcohol production. A third potential route involved alpha-ketoisovalerate reductase, but this had no role in the formation of isobutyl alcohol from alpha-hydroxyisovalerate because cell homogenates could not convert alpha-hydroxyisovalerate to isobutyl alcohol. The final possibility, use of the pyruvate decarboxylase-like enzyme encoded by YDL080c, seemed to be irrelevant, because a strain with a disruption in this gene produced wild-type levels of isobutyl alcohol. Thus there are major differences in the catabolism of leucine and valine to their respective "fusel" alcohols. Whereas in the catabolism of leucine to isoamyl alcohol the major route is via the decarboxylase encoded by YDL080c, any single isozyme of pyruvate decarboxylase is sufficient for the formation of isobutyl alcohol from valine. Finally, analysis of the 13C-labeled products revealed that the pathways of valine catabolism and leucine biosynthesis share a common pool of alpha-ketoisovalerate. << Less

  J. Biol. Chem. 273:25751-25756(1998) [PubMed] [EuropePMC]

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