The active (ADHa) and inactive (ADHi) forms of the PQQ-alcohol dehydrogenase from Gluconacetobacter diazotrophicus differ in their respective oligomeric structures and redox state of their corresponding prosthetic groups

Abstract

The membrane-bound alcohol dehydrogenase of Gluconacetobacter diazotrophicus contains one pyrroloquinoline quinone moiety (PQQ), one [2Fe-2S] cluster, and four c-type cytochromes. Here, we describe a novel and inactive enzyme. ADHi, similarly to ADHa, is a heterodimer of 72- and 44-kDa subunits and contains the expected prosthetic groups. However, ADHa showed a threefold molecular mass as compared to ADHi. Noteworthy, the PQQ, the [2Fe-2S] and most of the cytochromes in purified ADHi is in the oxidized form, contrasting with ADHa where the PQQ-semiquinone is detected and the [2Fe-2S] cluster as well as the cytochromes c remained fully reduced after purification. Reduction kinetics of the ferricyanide-oxidized enzymes showed that while ADHa was brought back by ethanol to its full reduction state, in ADHi, only one-quarter of the total heme c was reduced. The dithionite-reduced ADHi was largely oxidized by ubiquinone-2, thus indicating that intramolecular electron transfer is not impaired in ADHi. The acidic pH of the medium might be deleterious for the membrane-bound ADH by causing conformational changes leading to changes in the relative orientation of heme groups and shift of corresponding redox potential to higher values. This would hamper electron transfer resulting in the low activity observed in ADHi.

The Oxidative Fermentation of Ethanol in Gluconacetobacter diazotrophicus Is a Two-Step Pathway Catalyzed by a Single Enzyme: Alcohol-Aldehyde Dehydrogenase (ADHa)

“…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 5 A), the ADHa with an optimum pH 6.0 ( Figure 5 B), 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.…”

“…Our results suggest that at the beginning of the growth of Ga. diazoptrophicus (the first 5 to 10 h; Figure 5 A), the ADHa with an optimum pH 6.0 ( Figure 5 B), 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 5 A) 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 5 B). 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 ].…”

“…SKU 14 [ 17 ], Ga. diazotrophiocus [ 18 ] and Acetobacter pasteurianus MSU10 [ 19 ], ADH enzymes exhibited activity, also with formaldehyde and acetaldehyde. In previous studies Gomez-Manzo et al [ 18 , 20 ] purified and characterized two types of Class III ADHs from Ga. diazotrophicus : ADH active (ADHa) and the inactive (ADHi). Both quinohemoproteins have the same oligomeric and prosthetic group composition.…”

“…Both quinohemoproteins have the same oligomeric and prosthetic group composition. However, both enzymes showed differences in the ability to oxidize alcohol, where the ADHi was several fold less active than its active counterparts [ 14 , 20 ].…”

Physiology of Acetic Acid Bacteria and Their Role in Vinegar and Fermented Beverages

“…When these gluconobacters are grown in acidic or high aeration conditions, they produce a large amount of ADH protein, but ADH activity remains unchanged, suggesting the presence of an inactive protein. Such inactive ADH displays a tenth of the activity of the active form (Gomez‐Manzo et al., ; Matsushita, Yakushi, Takaki, Toyama, & Adachi, ). Certain cultivation conditions such as low pH and/or high aeration also reduce ADH activity, such as in Gluconobacter suboxydans , where low aeration was shown to favor active over inactive ADH formation (Matsushita et al., ).…”