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

ABSTRACT: 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 pdc1? pdc5? pdc6? aro10? thi3? 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.

SUBMITTER: Romagnoli G

PROVIDER: S-EPMC3485725 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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

Romagnoli Gabriele G Luttik Marijke A H MA Kötter Peter P Pronk Jack T JT Daran Jean-Marc JM

Applied and environmental microbiology 20120817 21

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 enc ...[more]

PMID: 22904058

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Project description:The irreversible decarboxylation step, which commits 2-oxo acids to the Ehrlich pathway, was initially attributed to pyruvate decarboxylase. However, the yeast genome was shown to harbour no fewer than 5 genes that show sequence similarity with thiamine-diphosphate dependent decarboxylase genes. Three of these (PDC1, PDC5 and PDC6) encode pyruvate decarboxylases { while ARO10 and THI3 represent alternative candidates for Ehrlich-pathway decarboxylases. Transcriptome analysis and decarboxylase activity measurements on an S. cerevisiae aro10 strain, a double aro10 thi3 deletion strain and a quadruple pdc1,5,6,aro10 mutant strains grown in carbonÃ¢â¬âlimited chemostat with phenylalanine as nitrogen source indicated that:; i) PDC5 is strongly upregulated in an aro10 background (Fig. 2) and also encodes a broad-substrate ÃÂ±-keto acid decarboxylase. ii) PDC5 expression depends on the presence of THI3 (Fig. 2), and; iii) in contrast to cell extracts from a strain expressing ARO10 only (pdc1,5,6, thi3), cell extract from a strain that only contains THI3 (pdc1,5,6,aro10) did not produce any ÃÂ±-keto acid decarboxylase activity . THI3 has recently been demonstrated to be involved in regulation of thiamine homeostasis in S. cerevisiae, which further suggests that its role in the Ehrlich pathway may be regulatory rather than catalytic. A systematic investigation of the catalytic properties of all five (putative) TPP-dependent decarboxylases (Aro10p, Thi3p, Pdc1p, Pdc5p, Pdc6p) is essential for a final resolution of the substrate specificity of these key enzymes in the Ehrlich pathway. Experiment Overall Design: Comparison of glucose limited chemostat cultivations with phenylalanine as sole nitrogen sources of the following strains: Experiment Overall Design: - CENPK113-7D MATa MAL2-8c SUC2 Experiment Overall Design: - CENPK555-4A MATa MAL2-8c SUC2 ydr380w delta Experiment Overall Design: - CENPK5632-3B MATalpha MAL2-8c SUC2 ydr380w delta ydl080c delta Experiment Overall Design: - CENPK609-11A MATa MAL2-8c SUC2 pdc1, 5, 6 delta ydr380w delta

Project description:BACKGROUND:Decarboxylation of ?-ketoisovalerate to isobutyraldehyde is a key reaction in metabolic engineering of Saccharomyces cerevisiae for isobutanol production with published studies relying on overexpression of either the native ARO10 gene or of the Lactococcus lactis kivD decarboxylase gene resulting in low enzymatic activities. Here, we compare relevant properties for isobutanol production of Aro10, KivD and an additional, less studied, L. lactis decarboxylase KdcA. RESULTS:To eliminate interference by native decarboxylases, each 2-oxo acid decarboxylase was overexpressed in a 'decarboxylase-negative' (pdc1? pdc5? pdc6? aro10?) S. cerevisiae background. Kinetic analyses in cell extracts revealed a superior V max/K m ratio of KdcA for ?-ketoisovalerate and a wide range of linear and branched-chain 2-oxo acids. However, KdcA also showed the highest activity with pyruvate which, in engineered strains, can contribute to formation of ethanol as a by-product. Removal of native decarboxylase genes eliminated growth on valine as sole nitrogen source and subsequent complementation of this growth impairment by expression of each decarboxylase indicated that based on the increased growth rate, the in vivo activity of KdcA with ?-ketoisovalerate was higher than that of KivD and Aro10. Moreover, during oxygen-limited incubation in the presence of glucose, strains expressing kdcA or kivD showed a ca. twofold higher in vivo rate of conversion of ?-ketoisovalerate into isobutanol than an ARO10-expressing strain. Finally, cell extracts from cultures grown on different nitrogen sources revealed increased activity of constitutively expressed KdcA after growth on both valine and phenylalanine, while KivD and Aro10 activity was only increased after growth on phenylalanine suggesting a difference in the regulation of these enzymes. CONCLUSIONS:This study illustrates important differences in substrate specificity, enzyme kinetics and functional expression between different decarboxylases in the context of isobutanol production and identifies KdcA as a promising alternative decarboxylase not only for isobutanol production but also for other branched-chain and linear alcohols.

Project description:BackgroundStandard numbering schemes for families of homologous proteins allow for the unambiguous identification of functionally and structurally relevant residues, to communicate results on mutations, and to systematically analyse sequence-function relationships in protein families. Standard numbering schemes have been successfully implemented for several protein families, including lactamases and antibodies, whereas a numbering scheme for the structural family of thiamine-diphosphate (ThDP) -dependent decarboxylases, a large subfamily of the class of ThDP-dependent enzymes encompassing pyruvate-, benzoylformate-, 2-oxo acid-, indolpyruvate- and phenylpyruvate decarboxylases, benzaldehyde lyase, acetohydroxyacid synthases and 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD) is still missing.Despite a high structural similarity between the members of the ThDP-dependent decarboxylases, their sequences are diverse and make a pairwise sequence comparison of protein family members difficult.ResultsWe developed and validated a standard numbering scheme for the family of ThDP-dependent decarboxylases. A profile hidden Markov model (HMM) was created using a set of representative sequences from the family of ThDP-dependent decarboxylases. The pyruvate decarboxylase from S. cerevisiae (PDB: 2VK8) was chosen as a reference because it is a well characterized enzyme. The crystal structure with the PDB identifier 2VK8 encompasses the structure of the ScPDC mutant E477Q, the cofactors ThDP and Mg(2+) as well as the substrate analogue (2S)-2-hydroxypropanoic acid. The absolute numbering of this reference sequence was transferred to all members of the ThDP-dependent decarboxylase protein family. Subsequently, the numbering scheme was integrated into the already established Thiamine-diphosphate dependent Enzyme Engineering Database (TEED) and was used to systematically analyze functionally and structurally relevant positions in the superfamily of ThDP-dependent decarboxylases.ConclusionsThe numbering scheme serves as a tool for the reliable sequence alignment of ThDP-dependent decarboxylases and the unambiguous identification and communication of corresponding positions. Thus, it is the basis for the systematic and automated analysis of sequence-encoded properties such as structural and functional relevance of amino acid positions, because the analysis of conserved positions, the identification of correlated mutations and the determination of subfamily specific amino acid distributions depend on reliable multisequence alignments and the unambiguous identification of the alignment columns. The method is reliable and robust and can easily be adapted to further protein families.

Dataset Information

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

Publications

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

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