Comparative characterisation of thiamin diphosphate-dependent decarboxylases

Gocke, Dörte; Graf, Thorsten; Brosi, Helen; Frindi-Wosch, Ilona; Walter, Lydia; Müller, Michael; Pohl, Martina

doi:10.1016/j.molcatb.2009.03.019

Cited by 43 publications

(52 citation statements)

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“…The pH optimum of GoxPDC was determined to lie between 4.5 and 5.0, similar to that of the PDC from A. pasteurianus and slightly lower than for other PDCs from Gram negative bacteria (Figure 3) (Gocke et al, 2009). The temperature optimum of GoxPDC was between 50 to 55°C.…”

Section: Kinetic Characterization Of the Goxpdc Enzymementioning

confidence: 53%

“…The substrate recognition and decarboxylation range of the enzyme is similar to that of other Gram negative PDCs , showing a preference for short-chain aliphatic 2-keto acids (Gocke et al, 2009). The value of k cat /K M for pyruvate compared to those for 2-ketobutanoate and 2-ketopentanoate, the nearest analogues, and the retention of Ile468, thought to be involved in substrate specificity, suggests that this enzyme favors pyruvate as its physiological substrate (Siegert et al, 2005;Gocke et al, 2009). In terms of its kinetic behavior, GoxPDC appears to behave in a manner similar to other Gram-negative bacterial PDCs, displaying the same pH dependent increase in k cat /K M while catalytic efficiency (k cat ) remains largely unchanged.…”

Section: Discussionmentioning

confidence: 65%

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Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

Zyl

Taylor

Eley

et al. 2013

Appl Microbiol Biotechnol

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This study reports the expression, purification and kinetic characterization of a PDC from Gluconobacter oxydans. Kinetic analyses showed the enzyme to have high affinity for pyruvate (120µM at pH 5), high catalytic efficiency (4.75 x 10 5 M -1 s -1 at pH 5), a pH opt of approximately 4.5 and an in vitro temperature optimum at approximately 55°C (the highest yet reported for a b a c t e r i a l P D C ) . D u e t o g o o d in vitro thermostablity (approximately 40% enzyme activity retained after 30 minutes at 65°C) this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius. Initial studies using a variety of methods failed to detect activity at any growth temperature. However, the application of codon harmonization (i.e., mimicry of the heterogeneous host's transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45°C (as determined by enzyme activity, Western blot, mRNA detection and ethanol productivity). Here we describe the successful expression of PDC in a true thermophile. Yields as high as 0.35 g/g ±0.04 ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights that the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription / translation coupling.

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Section: Kinetic Characterization Of the Goxpdc Enzymementioning

confidence: 53%

Section: Discussionmentioning

confidence: 65%

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

Zyl

Taylor

Eley

et al. 2013

Appl Microbiol Biotechnol

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“…With CDO as a substrate, the activity of CDH was highest at pH 8.0 (see Fig. S1 in the supplemental material), whereas lower pH values were reported for POX from L. plantarum (pH 5.7) (60) and various 2-ketoacid decarboxylases (20,41). CDH showed a relatively steep increase in specific activity in the pH range from 6.5 to 8.0, which might be related to the formation of the CDO monohydrate.…”

Section: Discussionmentioning

confidence: 98%

“…Recently, the catalytic properties of several ThDP-dependent 2-ketoacid decarboxylases have been compared. With pyruvate as a substrate, the bacterial enzymes exhibited similar kinetic parameters, with K m values in the range from 1.3 to 2.8 mM (20). Notably, CDH did not accept pyruvate as a substrate, and it did not react with compounds structurally related to either CDO or 6-oxohexanoate (18).…”

Section: Discussionmentioning

confidence: 99%

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Cyclohexane-1,2-Dione Hydrolase from Denitrifying Azoarcus sp. Strain 22Lin, a Novel Member of the Thiamine Diphosphate Enzyme Family

et al. 2011

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Alicyclic compounds with hydroxyl groups represent common structures in numerous natural compounds, such as terpenes and steroids. Their degradation by microorganisms in the absence of dioxygen may involve a COC bond ring cleavage to form an aliphatic intermediate that can be further oxidized. The cyclohexane-1,2-dione hydrolase (CDH) (EC 3.7.1.11) from denitrifying Azoarcus sp. strain 22Lin, grown on cyclohexane-1,2-diol as a sole electron donor and carbon source, is the first thiamine diphosphate (ThDP)-dependent enzyme characterized to date that cleaves a cyclic aliphatic compound. The degradation of cyclohexane-1,2-dione (CDO) to 6-oxohexanoate comprises the cleavage of a COC bond adjacent to a carbonyl group, a typical feature of reactions catalyzed by ThDP-dependent enzymes. In the subsequent NAD ؉ -dependent reaction, 6-oxohexanoate is oxidized to adipate. CDH has been purified to homogeneity by the criteria of gel electrophoresis (a single band at ϳ59 kDa; calculated molecular mass, 64.5 kDa); in solution, the enzyme is a homodimer (ϳ105 kDa; gel filtration). As isolated, CDH contains 0.8 ؎ 0.05 ThDP, 1.0 ؎ 0.02 Mg 2؉ , and 1.0 ؎ 0.015 flavin adenine dinucleotide (FAD) per monomer as a second organic cofactor, the role of which remains unclear. Strong reductants, Ti(III)-citrate, Na ؉ -dithionite, and the photochemical 5-deazaflavin/oxalate system, led to a partial reduction of the FAD chromophore. The cleavage product of CDO, 6-oxohexanoate, was also a substrate; the corresponding cyclic 1,3-and 1,4-diones did not react with CDH, nor did the cis-and trans-cyclohexane diols. The enzymes acetohydroxyacid synthase (AHAS) from Saccharomyces cerevisiae, pyruvate oxidase (POX) from Lactobacillus plantarum, benzoylformate decarboxylase from Pseudomonas putida, and pyruvate decarboxylase from Zymomonas mobilis were identified as the closest relatives of CDH by comparative amino acid sequence analysis, and a ThDP binding motif and a 2-fold Rossmann fold for FAD binding could be localized at the C-terminal end and central region of CDH, respectively. A first mechanism for the ring cleavage of CDO is presented, and it is suggested that the FAD cofactor in CDH is an evolutionary relict.Alicyclic compounds, such as steroids and terpenes, are widespread in nature. They are produced by plant cells as secondary metabolites and occur in fossil fuels. Microorganisms can convert these compounds to cellular metabolites under oxic and anoxic conditions. Their biodegradation proceeds via COC bond ring cleavage to form an aliphatic intermediate, which can be further degraded by ␤-oxidation. In aerobic bacteria, the cleavage of the cyclic compound is catalyzed by a NADPH-dependent, flavin-containing monooxygenase. For example, cyclohexanone is converted to ε-caprolactone in a Bayer-Villiger-type reaction (14). Subsequently, the lactone is hydrolyzed to 6-hydroxyhexanoate (63), followed by two NAD ϩ /NADP ϩ -dependent oxidation steps with adipate as the final product. In anaerobes, such as Pseudomonas sp. strain K601, cyclohexa...

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Comparative characterisation of thiamin diphosphate-dependent decarboxylases

Cited by 43 publications

References 43 publications

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

Cyclohexane-1,2-Dione Hydrolase from Denitrifying Azoarcus sp. Strain 22Lin, a Novel Member of the Thiamine Diphosphate Enzyme Family

Organic Synthesis with Lyases

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