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The oxidative fermentation of ethanol in Gluconacetobacter diazotrophicus is a two-step pathway catalyzed by a single enzyme: alcohol-aldehyde Dehydrogenase (ADHa).

Gómez-Manzo S, Escamilla JE, González-Valdez A, López-Velázquez G, Vanoye-Carlo A, Marcial-Quino J, de la Mora-de la Mora I, Garcia-Torres I, Enríquez-Flores S, Contreras-Zentella ML, Arreguín-Espinosa R, Kroneck PM, Sosa-Torres ME - Int J Mol Sci (2015)

Bottom Line: The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH).We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2-C6) and its respective aldehydes as alternate substrates.Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, S.S. Mexico City 04530, Mexico. saulmanzo@ciencias.unam.mx.

ABSTRACT
Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2-C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde.

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Determination of acetaldehyde-semicarbazone complex as the final product of the ADHa. (A) UV/vis spectra of the formation of acetaldehyde-semicarbazone complex at 223 nm. Spectra was recorded at three min intervals; (B) Determination of the acetaldehyde released during the reaction by the enzyme ADHa and trapped by the semicarbazide; (C) Time course of ethanol and (D) allylic alcohol oxidation by the ADHa from Ga. diazotrophicus.
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ijms-16-01293-f004: Determination of acetaldehyde-semicarbazone complex as the final product of the ADHa. (A) UV/vis spectra of the formation of acetaldehyde-semicarbazone complex at 223 nm. Spectra was recorded at three min intervals; (B) Determination of the acetaldehyde released during the reaction by the enzyme ADHa and trapped by the semicarbazide; (C) Time course of ethanol and (D) allylic alcohol oxidation by the ADHa from Ga. diazotrophicus.

Mentions: To corroborate the results obtained using radioactive [1-14C]ethanol, the same test was performed under the same experimental conditions using ethanol HPLC grade. The formation of the acetaldehyde-semicarbazone complex was measured during time in hermetically closed cuvettes (Figure 4A). As seen, the absorbance spectrum of the semicarbazide showed a shift in signal from semicarbazide to semicarbazone (223 nm) as showed in the Figure 4A. According to the signal intensity and using the calibration curve with acetaldehyde-semicarbazone, we determined that the concentration of acetaldehyde released during the reaction by the enzyme ADHa and trapped by the semicarbazide was 0.5 mM (Figure 4B); which corresponds to 2.3% of the final product, when the reaction was started with an excess of ethanol (100 mM). However, if we consider the specificity constant (kcat) and the reaction time in this assay, we calculate that of the 100 mM of initial substrate, only 25 mM ethanol has been catalyzed to product by ADHa in 24 min. Thus, from 100% of product (25 mM), 97.3% of the oxidized ethanol is brought to acetate form and only 2% (0.5 mM) is released as acetaldehyde.


The oxidative fermentation of ethanol in Gluconacetobacter diazotrophicus is a two-step pathway catalyzed by a single enzyme: alcohol-aldehyde Dehydrogenase (ADHa).

Gómez-Manzo S, Escamilla JE, González-Valdez A, López-Velázquez G, Vanoye-Carlo A, Marcial-Quino J, de la Mora-de la Mora I, Garcia-Torres I, Enríquez-Flores S, Contreras-Zentella ML, Arreguín-Espinosa R, Kroneck PM, Sosa-Torres ME - Int J Mol Sci (2015)

Determination of acetaldehyde-semicarbazone complex as the final product of the ADHa. (A) UV/vis spectra of the formation of acetaldehyde-semicarbazone complex at 223 nm. Spectra was recorded at three min intervals; (B) Determination of the acetaldehyde released during the reaction by the enzyme ADHa and trapped by the semicarbazide; (C) Time course of ethanol and (D) allylic alcohol oxidation by the ADHa from Ga. diazotrophicus.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307304&req=5

ijms-16-01293-f004: Determination of acetaldehyde-semicarbazone complex as the final product of the ADHa. (A) UV/vis spectra of the formation of acetaldehyde-semicarbazone complex at 223 nm. Spectra was recorded at three min intervals; (B) Determination of the acetaldehyde released during the reaction by the enzyme ADHa and trapped by the semicarbazide; (C) Time course of ethanol and (D) allylic alcohol oxidation by the ADHa from Ga. diazotrophicus.
Mentions: To corroborate the results obtained using radioactive [1-14C]ethanol, the same test was performed under the same experimental conditions using ethanol HPLC grade. The formation of the acetaldehyde-semicarbazone complex was measured during time in hermetically closed cuvettes (Figure 4A). As seen, the absorbance spectrum of the semicarbazide showed a shift in signal from semicarbazide to semicarbazone (223 nm) as showed in the Figure 4A. According to the signal intensity and using the calibration curve with acetaldehyde-semicarbazone, we determined that the concentration of acetaldehyde released during the reaction by the enzyme ADHa and trapped by the semicarbazide was 0.5 mM (Figure 4B); which corresponds to 2.3% of the final product, when the reaction was started with an excess of ethanol (100 mM). However, if we consider the specificity constant (kcat) and the reaction time in this assay, we calculate that of the 100 mM of initial substrate, only 25 mM ethanol has been catalyzed to product by ADHa in 24 min. Thus, from 100% of product (25 mM), 97.3% of the oxidized ethanol is brought to acetate form and only 2% (0.5 mM) is released as acetaldehyde.

Bottom Line: The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH).We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2-C6) and its respective aldehydes as alternate substrates.Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, S.S. Mexico City 04530, Mexico. saulmanzo@ciencias.unam.mx.

ABSTRACT
Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2-C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde.

Show MeSH
Related in: MedlinePlus