Gene Cloning and Biochemical Characterization of an Alcohol Dehydrogenase from Euglena gracilis¹

Abstract

Euglena gracilis is a freshwater free-living organism able to grow with ethanol as carbon source; to facilitate this metabolism several alcohol dehydrogenase (ADH) activities have been detected. We report the gene cloning, over-expression, and biochemical characterization of a medium-chain NAD(+)-dependent ADH from E. gracilis (EgADH). The enzyme's amino acid sequence displayed the highest percentages of similarity and identity with ADHs of bacteria and fungi. In the predicted three-dimensional model, all the residues involved in Zn(2+), cofactor, and substrate binding were conserved. A conventional signal peptide for import into mitochondria could not be clearly identified. The protein of 37 kDa was over-expressed, purified to homogeneity, and kinetically characterized. The enzyme's optimal pH was 7.0 for ethanol oxidation displaying a V(m) of 11.7+/-3.6 U/mg protein and a K(m) of 3.2+/-0.7 mM for this substrate. Isopropanol and isopentanol were also utilized, although with less efficiency. It showed specificity for NAD(+) with a K(m) value of 0.39+/-0.1 mM and Mg(2+) or Zn(2+) were essential for activity. The recombinant EgADH reported here may help to elucidate the roles that different ADHs have on the metabolism of short- and long-chain alcohols in this microorganism.

The Mitochondrion of Euglena gracilis

“…Several alcohol dehydrogenases (ADHs) have been reported to localize to the cytosol (Munir et al 2002;Palma-Gutiérrez et al 2008), whereas prior work had identified ADHs only in Euglena mitochondria (Ono et al 1995a). Furthermore, one aldehyde dehydrogenase (ALDH) (Ono et al 1995b;Rodríguez-Zavala et al 2006) and one acetyl-CoA synthase (Ono et al 1995a) were found in the mitochondria.…”

Comparative transcriptome analysis reveals different adaptation mechanisms for degradation of very long‐chain and normal long‐chain alkanes in Dietzia sp. DQ12‐45‐1b

“…The third step in alkane degradation involves the conversion of the primary alcohol to its corresponding aldehyde then followed by conversion of the aldehyde to fatty acid, which are carried out by alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs), respectively (Ludwig et al ., 1995; Ishige et al ., 2000; Peng et al ., 2006; Palma‐Gutierrez et al ., 2008; Liu et al ., 2009; Kawano et al ., 2011). We identified eight ADHs homologues and five ALDHs homologues in DQ12‐45‐1b genome through multiple sequence alignment or phylogenetic analysis of ADH or ALDH related homologues from alkane‐degrading bacteria (Supporting Information Figs.…”