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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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(A) Growth properties of Ga. diazotrophicus in LGIP medium. (○) Determination of growth by optical density O.D.600nm and (●) Measurement of pH of the culture medium; (B) Quantification of reductase activity pH dependent properties of the purified membrane-bound Alcohol dehydrogenase active (ADHa), Alcohol dehydrogenase inactive (ADHi) and aldehyde dehydrogenase (ALDH) complexes purified from Ga. diazotrophicus. The ferricyanide reductase activity was measured in Mcllvaine buffer at different pH. The conditions used for the activity assays are described in Experimental Setion.
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ijms-16-01293-f005: (A) Growth properties of Ga. diazotrophicus in LGIP medium. (○) Determination of growth by optical density O.D.600nm and (●) Measurement of pH of the culture medium; (B) Quantification of reductase activity pH dependent properties of the purified membrane-bound Alcohol dehydrogenase active (ADHa), Alcohol dehydrogenase inactive (ADHi) and aldehyde dehydrogenase (ALDH) complexes purified from Ga. diazotrophicus. The ferricyanide reductase activity was measured in Mcllvaine buffer at different pH. The conditions used for the activity assays are described in Experimental Setion.

Mentions: Finally, taking into account the optimum pH previously reported for the membrane-bound active alcohol dehydrogenase (ADHa) [18], inactive alcohol dehydrogenase (ADHi; 15% of activity in respect to the active ADH) [20], and aldehyde dehydrogenase (ALDH) [25] from Ga. diazotrophicus, we propose that under physiological conditions, the bifunctional ADHa would permit the massive conversion of ethanol to acetic acid, usually seen in the acetic acid bacteria, without the inconvenient transient accumulation of the highly toxic acetaldehyde. Our results suggest that at the beginning of the growth of Ga. diazoptrophicus (the first 5 to 10 h; Figure 5A), the ADHa with an optimum pH 6.0 (Figure 5B), might be able perform a rapid oxidation from ethanol to acetic acid present in the medium, and that these substrates are subjected to a two-step oxidation to produce acetic acid without releasing the acetaldehyde intermediary to the media. At the end phase of the growth (pH 3.5, after 30 to 40 h; Figure 5A) the ADHi, with an optimum pH of 4 [20] might be able to oxidize the small quantity of alcohol remaining in the culture medium, and the ALDH (optimum pH 3.5) would convert the acetaldehyde released in the media to acetate (Figure 5B). These data are in concordance with the aldehyde-ferricyanide reductase activity in native membranes of Ga. diazotrophicus which exhibited an optimum pH of 3.5 [25]. Therefore the results here indicate that the optimal pH determined for the ferricyanide reductase activity both in membranes and purified ALDH enzyme [25] is similar to the pH range at which acetic acid bacteria usually produce vinegar.


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)

(A) Growth properties of Ga. diazotrophicus in LGIP medium. (○) Determination of growth by optical density O.D.600nm and (●) Measurement of pH of the culture medium; (B) Quantification of reductase activity pH dependent properties of the purified membrane-bound Alcohol dehydrogenase active (ADHa), Alcohol dehydrogenase inactive (ADHi) and aldehyde dehydrogenase (ALDH) complexes purified from Ga. diazotrophicus. The ferricyanide reductase activity was measured in Mcllvaine buffer at different pH. The conditions used for the activity assays are described in Experimental Setion.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-01293-f005: (A) Growth properties of Ga. diazotrophicus in LGIP medium. (○) Determination of growth by optical density O.D.600nm and (●) Measurement of pH of the culture medium; (B) Quantification of reductase activity pH dependent properties of the purified membrane-bound Alcohol dehydrogenase active (ADHa), Alcohol dehydrogenase inactive (ADHi) and aldehyde dehydrogenase (ALDH) complexes purified from Ga. diazotrophicus. The ferricyanide reductase activity was measured in Mcllvaine buffer at different pH. The conditions used for the activity assays are described in Experimental Setion.
Mentions: Finally, taking into account the optimum pH previously reported for the membrane-bound active alcohol dehydrogenase (ADHa) [18], inactive alcohol dehydrogenase (ADHi; 15% of activity in respect to the active ADH) [20], and aldehyde dehydrogenase (ALDH) [25] from Ga. diazotrophicus, we propose that under physiological conditions, the bifunctional ADHa would permit the massive conversion of ethanol to acetic acid, usually seen in the acetic acid bacteria, without the inconvenient transient accumulation of the highly toxic acetaldehyde. Our results suggest that at the beginning of the growth of Ga. diazoptrophicus (the first 5 to 10 h; Figure 5A), the ADHa with an optimum pH 6.0 (Figure 5B), might be able perform a rapid oxidation from ethanol to acetic acid present in the medium, and that these substrates are subjected to a two-step oxidation to produce acetic acid without releasing the acetaldehyde intermediary to the media. At the end phase of the growth (pH 3.5, after 30 to 40 h; Figure 5A) the ADHi, with an optimum pH of 4 [20] might be able to oxidize the small quantity of alcohol remaining in the culture medium, and the ALDH (optimum pH 3.5) would convert the acetaldehyde released in the media to acetate (Figure 5B). These data are in concordance with the aldehyde-ferricyanide reductase activity in native membranes of Ga. diazotrophicus which exhibited an optimum pH of 3.5 [25]. Therefore the results here indicate that the optimal pH determined for the ferricyanide reductase activity both in membranes and purified ALDH enzyme [25] is similar to the pH range at which acetic acid bacteria usually produce vinegar.

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