Effects of Metal Ions, Thiamine Diphosphate Analogues and Subunit Interactions on the Reconstitution Behaviour of Pyruvate Decarboxylase from Brewer’s Yeast

Eppendorfer, Steffen; König, Stephan; Golbik, Ralph; Neef, H; Lehle, Karla; Jaenicke, Rainer; Schellenberger, Alfred; Hübner, Gerhard

doi:10.1515/bchm3.1993.374.7-12.1129

Cited by 17 publications

(8 citation statements)

References 6 publications

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“…The hyperbolic dependence of the rate constants of reconstitution, calculated from the corresponding progress curves, on the concentration of ThDP (Fig. 6 inset) is indicative of a two-step mechanism of cofactor binding as described previously by Schellenberger and Hu¨bner [27] and Eppendorfer et al [42] for ScPDC. The reconstitution starts with binding of ThDP and Mg 2+ to the apoenzyme, followed by a conformational change to the catalytically active holoenzyme.…”

Section: Discussionsupporting

confidence: 74%

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Studies on structure–function relationships of indolepyruvate decarboxylase from Enterobacter cloacae, a key enzyme of the indole acetic acid pathway

Schütz

Golbik

Tittmann

et al. 2003

European Journal of Biochemistry

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Enterobacter cloacae, isolated from the rhizosphere of cucumbers, produces large amounts of indole‐3‐acetic acid. Indolepyruvate decarboxylase, the key enzyme in the biosynthetic pathway of indole‐3‐acetic acid, catalyses the formation of indole‐3‐acetaldehyde and carbon dioxide from indole‐3‐pyruvic acid. The enzyme requires the cofactors thiamine diphosphate and magnesium ions for catalytic activity. Recombinant indolepyruvate decarboxylase was purified from the host Escherichia coli strain JM109. Specificity of the enzyme for the substrates indole‐3‐pyruvic acid, pyruvic acid, benzoylformic acid, and seven benzoylformic acid analogues was investigated using a continuous optical assay. Stopped‐flow kinetic data showed no indication for substrate activation in the decarboxylation reaction of indole‐3‐pyruvic acid, pyruvic acid or benzoylformic acid. Size exclusion chromatography and small angle X‐ray solution scattering experiments suggested the tetramer as the catalytically active state and a pH‐dependent subunit association equilibrium. Analysis of the kinetic constants of the benzoylformic acid analogues according to Hansch et al. [Hansch, C., Leo, A., Unger, S.H., Kim, K.H., Nikaitani, D & Lien, E.J. (1973) J. Med. Chem.16, 1207–1216] and comparison with indole‐3‐pyruvic acid conversion by pyruvate decarboxylases from Saccharomyces cerevisiae and Zymomonas mobilis provided some insight into the catalytic mechanism of indolepyruvate decarboxylase.

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Section: Discussionsupporting

confidence: 74%

“…6 inset) is indicative of a two‐step mechanism of cofactor binding as described previously by Schellenberger and Hübner [27] and Eppendorfer et al . [42] for ScPDC. The reconstitution starts with binding of ThDP and Mg 2+ to the apoenzyme, followed by a conformational change to the catalytically active holoenzyme.…”

Section: Discussionmentioning

confidence: 99%

Studies on structure–function relationships of indolepyruvate decarboxylase from Enterobacter cloacae, a key enzyme of the indole acetic acid pathway

Schütz

Golbik

Tittmann

et al. 2003

European Journal of Biochemistry

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“…Hence, methylation at the N1′‐atom of the aminopyrimidine moiety mimics protonation of the enzyme‐bound cofactor by Glu418, which is specially required for its maximum activation in Sc Tk. However, the N1′‐methylated analogue of ThDP was found to be coenzymatically inactive in pyruvate decarboxylase from yeast [9,44]. We assume that reaction‐selected charge effects are responsible for these differences.…”

Section: Discussionmentioning

confidence: 99%

Effect of coenzyme modification on the structural and catalytic properties of wild‐type transketolase and of the variant E418A from Saccharomyces cerevisiae

et al. 2005

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Transketolase from baker's yeast is a thiamin diphosphate-dependent enzyme in sugar metabolism that reconstitutes with various analogues of the coenzyme. The methylated analogues (4¢-methylamino-thiamin diphosphate and N1¢-methylated thiamin diphosphate) of the native cofactor were used to investigate the function of the aminopyrimidine moiety of the coenzyme in transketolase catalysis. For the wild-type transketolase complex with the 4¢-methylamino analogue, no electron density was found for the methyl group in the X-ray structure, whereas in the complex with the N1¢-methylated coenzyme the entire aminopyrimidine ring was disordered. This indicates a high flexibility of the respective parts of the enzyme-bound thiamin diphosphate analogues. In the E418A variant of transketolase reconstituted with N1¢-methylated thiamin diphosphate, the electron density of the analogue was well defined and showed the typical V-conformation found in the wild-type holoenzyme [Lindqvist Y, Schneider G, Ermler U, Sundstrøm M (1992) EMBO J 11, 2373-2379]. The near-UV CD spectrum of the variant E418A reconstituted with N1¢-methylated thiamin diphosphate was identical to that of the wild-type holoenzyme, while the CD spectrum of the variant combined with the unmodified cofactor did not overlap with that of the native protein. The activation of the analogues was measured by the H ⁄ D-exchange at C2. Methylation at the N1¢ position of the cofactor activated the enzyme-bound cofactor analogue (as shown by a fast H ⁄ D-exchange rate constant). The absorbance changes in the course of substrate turnover of the different complexes investigated (transient kinetics) revealed the stability of the a-carbanion ⁄ enamine as the key intermediate in cofactor action to be dependent on the functionality of the 4-aminopyrimidine moiety of thiamin diphosphate.

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“…The physiological demand for Mg is greater than that for Ca 102 and an increased concentration of wort Ca is likely to exacerbate Mg deficiency through antagonistic interactions 172,174,197 , particularly since Ca may replace Mg in a number of biochemical pathways to the general detriment of the cell. Pyruvate decarboxylase activity, for example, is reduced when Ca is substituted in place of Mg 67 . Ca supplementation of wort may only be beneficial under certain circumstances, e.g., when wort Ca concentration is limited by the Ca con-tent of the local water supply.…”

Section: Calciummentioning

confidence: 99%

125th Anniversary Review: Improvement of Higher Gravity Brewery Fermentation via Wort Enrichment and Supplementation

Gibson

2011

Journal of the Institute of Brewing

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Intensification of the industrial brewing process, particularly the use of higher gravity worts, has been driven by increasing competition within the industry as well as the need to maximise the use of raw materials and minimise energy expenditure. These developments have, however, placed greater demands on brewing yeast strains, whose evolutionary history has not prepared them for the extreme conditions associated with higher gravity brewing. Various yeast nutrient supplements have been used or proposed to maintain yeast performance under stressful conditions. These have included specific metal ions, lipids and lipid components such as fatty acids and sterols and free amino nitrogen, usually supplied in the form of a complex yeast food. Correction of wort nutritional deficiencies may reduce stress sensitivity of yeast and improve fermentation performance. Potential negative consequences of altering wort composition must however be considered, as important beer quality attributes such as taste, stability and foam can be affected. Here, the various options for nutrient supplementation and their influence on yeast physiology and performance, as well as beer characteristics are considered.

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Effects of Metal Ions, Thiamine Diphosphate Analogues and Subunit Interactions on the Reconstitution Behaviour of Pyruvate Decarboxylase from Brewer’s Yeast

Cited by 17 publications

References 6 publications

Studies on structure–function relationships of indolepyruvate decarboxylase from Enterobacter cloacae, a key enzyme of the indole acetic acid pathway

Studies on structure–function relationships of indolepyruvate decarboxylase from Enterobacter cloacae, a key enzyme of the indole acetic acid pathway

Effect of coenzyme modification on the structural and catalytic properties of wild‐type transketolase and of the variant E418A from Saccharomyces cerevisiae

125th Anniversary Review: Improvement of Higher Gravity Brewery Fermentation via Wort Enrichment and Supplementation

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