Structure-function relationships and flexible tetramer assembly in pyruvate decarboxylase revealed by analysis of crystal structures

Furey, W.; Arjunan, Palaniappa; Chen, L.; Sax, M.; Guo, Feng; Jordan, Frank

doi:10.1016/s0167-4838(98)00073-9

Cited by 32 publications

(30 citation statements)

References 38 publications

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“…In pdc1‐8 Asp291 was substituted for asparagine and in pdc1‐14 Ser455 was replaced by phenylalanine (Table 2). Asp291 is located in a loop region in the β‐domain; Ser455 is located within a long helix within the γ‐domain, which also includes the well‐conserved signature motif of ThDP‐dependent enzymes [17,19,22,37,38].…”

Section: Resultsmentioning

confidence: 99%

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Autoregulation of yeast pyruvate decarboxylase gene expression requires the enzyme but not its catalytic activity

Eberhardt

Cederberg

et al. 1999

European Journal of Biochemistry

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In the yeast, Saccharomyces cerevisiae, pyruvate decarboxylase (Pdc) is encoded by the two isogenes PDC1 and PDC5. Deletion of the more strongly expressed PDC1 gene stimulates the promoter activity of both PDC1 and PDC5, a phenomenon called Pdc autoregulation. Hence, pdc1D strains have high Pdc specific activity and can grow on glucose medium. In this work we have characterized the mutant alleles pdc1-8 and pdc1-14, which cause strongly diminished Pdc activity and an inability to grow on glucose. Both mutant alleles are expressed as detectable proteins, each of which differs from the wild-type by a single amino acid. The cloned pdc1-8 and pdc1-14 alleles, as well as the in-vitro-generated pdc1-51 (Glu51Ala) allele, repressed expression of PDC5 and diminished Pdc specific activity. Thus, the repressive effect of Pdc1p on PDC5 expression seems to be independent of its catalytic activity. A pdc1-8 mutant was used to isolate spontaneous suppressor mutations, which allowed expression of PDC5. All three mutants characterized had additional mutations within the pdc1-8 allele. Two of these mutations resulted in a premature translational stop conferring phenotypes virtually indistinguishable from those of a pdc1D mutation. The third mutation, pdc1-803, led to a deletion of two amino acids adjacent to the pdc1-8 mutation. The alleles pdc1-8 and pdc1-803 were expressed in Escherichia coli and purified to homogeneity. In the crude extract, both proteins had 10% residual activity, which was lost during purification, probably due to dissociation of the cofactor thiamin diphosphate (ThDP). The defect in pdc1-8 (Asp291Asn) and the two amino acids deleted in pdc1-803 (Ser296 and Phe297) are located within a flexible loop in the b domain. This domain appears to determine the relative orientation of the a and g domains, which bind ThDP. Alterations in this loop may also affect the conformational change upon substrate binding. The mutation in pdc1-14 (Ser455Phe) is located within the ThDP fold and is likely to affect binding and/or orientation of the cofactor in the protein. We suggest that autoregulation is triggered by a certain conformation of Pdc1p and that the mutations in pdc1-8 and pdc1-14 may lock Pdc1p in vivo in a conformational state which leads to repression of PDC5.

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Section: Resultsmentioning

confidence: 99%

“…The structure–function relationship of Pdc has been studied intensively and the three‐dimensional structure of the protein has been resolved from two different types of crystals [17–19]. The protein is a homotetramer, which readily dissociates into very stable dimers: each subunit consists of three domains of approximately equal size: α, β and γ.…”

mentioning

confidence: 99%

Autoregulation of yeast pyruvate decarboxylase gene expression requires the enzyme but not its catalytic activity

Eberhardt

Cederberg

et al. 1999

European Journal of Biochemistry

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“…However, the poor correlation between the transcript level and enzyme activity (Table 5) indicates that posttranscriptional regulation of the decarboxylase activities also occurs. An attractive model for this posttranscriptional regulation depends upon the in vivo tetrameric form of pyruvate decarboxylase (21). If the Pdc-like proteins can form heterotetramers, the resulting decarboxylase activities may have different substrate specificities.…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of Phenylpyruvate Decarboxylase Genes in Saccharomyces cerevisiae

Vuralhan

Morais

Tai

et al. 2003

Appl Environ Microbiol

168

159

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Catabolism of amino acids via the Ehrlich pathway involves transamination to the corresponding ␣-keto acids, followed by decarboxylation to an aldehyde and then reduction to an alcohol. Alternatively, the aldehyde may be oxidized to an acid. This pathway is functional in Saccharomyces cerevisiae, since during growth in glucose-limited chemostat cultures with phenylalanine as the sole nitrogen source, phenylethanol and phenylacetate were produced in quantities that accounted for all of the phenylalanine consumed. Our objective was to identify the structural gene(s) required for the decarboxylation of phenylpyruvate to phenylacetaldehyde, the first specific step in the Ehrlich pathway. S. cerevisiae possesses five candidate genes with sequence similarity to genes encoding thiamine diphosphate-dependent decarboxylases that could encode this activity: YDR380w/ ARO10, YDL080C/THI3, PDC1, PDC5, and PDC6. Phenylpyruvate decarboxylase activity was present in cultures grown with phenylalanine as the sole nitrogen source but was absent from ammonia-grown cultures. Furthermore, the transcript level of one candidate gene (ARO10) increased 30-fold when phenylalanine replaced ammonia as the sole nitrogen source. Analyses of phenylalanine catabolite production and phenylpyruvate decarboxylase enzyme assays indicated that ARO10 was sufficient to encode phenylpyruvate decarboxylase activity in the absence of the four other candidate genes. There was also an alternative activity with a higher capacity but lower affinity for phenylpyruvate. The candidate gene THI3 did not itself encode an active phenylpyruvate decarboxylase but was required along with one or more pyruvate decarboxylase genes (PDC1, PDC5, and PDC6) for the alternative activity. The K m and V max values of the two activities differed, showing that Aro10p is the physiologically relevant phenylpyruvate decarboxylase in wild-type cells. Modifications to this gene could therefore be important for metabolic engineering of the Ehrlich pathway.

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“…So far, only two types of overall tetramer crystal structures of yeast PDCs are known, the open (37) and half-side closed (28,38,39) conformation (Fig. 3).…”

Section: Activator Binding Studies Using Saxsmentioning

confidence: 99%

Covalently Bound Substrate at the Regulatory Site of Yeast Pyruvate Decarboxylases Triggers Allosteric Enzyme Activation

Kutter

Weiss

Wille

et al. 2009

Journal of Biological Chemistry

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The mechanism by which the enzyme pyruvate decarboxylase from two yeast species is activated allosterically has been elucidated. A total of seven three-dimensional structures of the enzyme, of enzyme variants, or of enzyme complexes from two yeast species, three of them reported here for the first time, provide detailed atomic resolution snapshots along the activation coordinate. The prime event is the covalent binding of the substrate pyruvate to the side chain of cysteine 221, thus forming a thiohemiketal. This reaction causes the shift of a neighboring amino acid, which eventually leads to the rigidification of two otherwise flexible loops, one of which provides two histidine residues necessary to complete the enzymatically competent active site architecture. The structural data are complemented and supported by kinetic investigations and binding studies, providing a consistent picture of the structural changes occurring upon enzyme activation.

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Structure-function relationships and flexible tetramer assembly in pyruvate decarboxylase revealed by analysis of crystal structures

Cited by 32 publications

References 38 publications

Autoregulation of yeast pyruvate decarboxylase gene expression requires the enzyme but not its catalytic activity

Autoregulation of yeast pyruvate decarboxylase gene expression requires the enzyme but not its catalytic activity

Identification and Characterization of Phenylpyruvate Decarboxylase Genes in Saccharomyces cerevisiae

Covalently Bound Substrate at the Regulatory Site of Yeast Pyruvate Decarboxylases Triggers Allosteric Enzyme Activation

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