Molecular and Catalytic Properties of the Aldehyde Dehydrogenase of Gluconacetobacter diazotrophicus , a Quinoheme Protein Containing Pyrroloquinoline Quinone, Cytochrome b , and Cytochrome c

Abstract

Several aldehyde dehydrogenase (ALDH) complexes have been purified from the membranes of acetic acid bacteria. The enzyme structures and the chemical nature of the prosthetic groups associated with these enzymes remain a matter of debate. We report here on the molecular and catalytic properties of the membrane-bound ALDH complex of the diazotrophic bacterium Gluconacetobacter diazotrophicus. The purified ALDH complex is a heterodimer comprising two subunits of 79.7 and 50 kDa, respectively. Reversed-phase high-pressure liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy led us to demonstrate, for the first time, the unequivocal presence of a pyrroloquinoline quinone prosthetic group associated with an ALDH complex from acetic acid bacteria. In addition, heme b was detected by UV-visible light (UV-Vis) spectroscopy and confirmed by reversed-phase HPLC. The smaller subunit bears three cytochromes c. Aliphatic aldehydes, but not formaldehyde, were suitable substrates. Using ferricyanide as an electron acceptor, the enzyme showed an optimum pH of 3.5 that shifted to pH 7.0 when phenazine methosulfate plus 2,6-dichlorophenolindophenol were the electron acceptors. Acetaldehyde did not reduce measurable levels of the cytochrome b and c centers; however, the dithionite-reduced hemes were conveniently oxidized by ubiquinone-1; this finding suggests that cytochrome b and the cytochromes c constitute an intramolecular redox sequence that delivers electrons to the membrane ubiquinone.

An Efficient Method Using Gluconacetobacter europaeus To Reduce an Unfavorable Flavor Compound, Acetoin, in Rice Vinegar Production

“…Several unique AAB are involved in cellulose production (2,3), symbiosis with insects (4), nitrogen fixing in the rhizosphere (5), and chronic granulomatous disease (6). Gluconacetobacter europaeus is representative of vinegar producers and is used for the production of high-acidity vinegar (acid concentration, Ͼ10%) in submerged bioreactors because it has extremely strong ethanol-oxidizing ability and ethanol/acetic acid resistance compared with other species (7).…”

Purification and Characterization of the Membrane-Bound Quinoprotein Glucose Dehydrogenase of Gluconacetobacter diazotrophicus PAL 5

“…In both cases, a signal was observed at 357 nm, suggesting the presence of PQQ in Ga. diazotrophicus GDHm. The signal at 357 nm is a characteristic signal previously reported in diverse quinoproteins and quinohemoproteins [5,[23][24][25]29]. The presence of PQQ was corroborated spectrofluorometrically of a methanol extract from GDHm (Fig.…”

“…After that, the extracted PQQ and the standards quinones were analyzed by RP-HPLC (Waters model 996). The prosthetic group was separated and identified in a RP-analytical column (Waters C18 Spherisorb S5 OD52; 4.6 9 150 mm) as described [24][25][26]. The system was calibrated using the following commercial standards: methanol-extracted PQQ associated with the purified ADHa and ALDH from Ga. diazotrophicus (retention times: 4.5 and 6.9 min, respectively).…”

“…For total protein, the gels were stained with silver or Coomassie Brilliant Blue R-250. The isoelectric point (pI) of the purified GDHm from Ga. diazotrophicus was performed according with the previously reported by Gomez-Manzo et al [23,25], in which a Phast system (Amersham Biosciences) and gels with a pH range of 3-9 were used.…”

Enhancement of rice vinegar production by modified semi-continuous culture based on analysis of enzymatic kinetic

“…Some works have revealed molecular properties and kinetic mechanism of PQQ-ADH which functions as the primary dehydrogenase in the ethanol oxidation respiratory chain, while PQQ-ALDH was not clear until now [18,19]. Then it is important to investigate the role and mechanism of PQQ-ALDH which seemed the rate-limiting enzyme because of its higher K m [20].…”