Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2° Adh) as a bifunctional alcohol dehydrogenase-acetyl-CoA reductive thioesterase

Abstract

The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described. The secondary-alcohol dehydrogenase (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa. The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation. The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation. The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose. The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling.

Respiratory behaviour of a Zymomonas mobilis adhB::kanr mutant supports the hypothesis of two alcohol dehydrogenase isoenzymes catalysing opposite reactions

“…Special functions for particular ADH isoenzymes might be common among ethanologenic microorganisms. An interesting scheme of two alcohol dehydrogenases catalysing opposite reactions has been postulated by Burdette and Zeikus [22,23] for anaerobically growing Thermoanaerobacter ethanolicus. They suggested that one of the two ADH isoenzymes in this anaerobe was participating both in ethanol synthesis from acetaldehyde and NADH, and in ethanol oxidation coupled to NADP + reduction, thus maintaining the redox balance between the NAD(P)(H) cofactor pools.…”

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

“…Resequencing was conducted essentially as described (27). Briefly, genomic DNA from C. thermocellum WT ATCC 27405 or EA mutant cultures was sent to the NimbleGen facility for CGS service following the company's procedure.…”

A multisubunit membrane-bound [NiFe] hydrogenase and an NADH-dependent Fe-only hydrogenase in the fermenting bacterium Thermoanaerobacter tengcongensis

“…ethanolicus, which produces ethanol as the major fermentation product. From this organism, a primary and a so-called secondary ADH have been purified and characterized (Burdette et al, 1996;Burdette & Zeikus, 1994). Both enzymes utilize a broad range of alcohols as substrates, but the enzymes can be clearly distinguished by their cofactor and substrate preferences.…”