Gene cloning and biochemical characterization of an alcohol dehydrogenase from Euglena gracilis.
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.
Palma-Gutiérrez
Helga N
HN
Departamento de Bioquímica, Instituto Nacional de Cardiología, Tlalpan, México D.F. 14080, México.
Rodríguez-Zavala
José Salud
JS
Jasso-Chávez
Ricardo
R
Moreno-Sánchez
Rafael
R
Saavedra
Emma
E
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005056:Euglena gracilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D015800:Protozoan Proteins; D000426:Alcohol Dehydrogenase