Premium
Gene Cloning and Biochemical Characterization of an Alcohol Dehydrogenase from Euglena gracilis 1
Author(s) -
PALMAGUTIÉRREZ HELGA N.,
RODRÍGUEZZAVALA JOSÉ SALUD,
JASSOCHÁVEZ RICARDO,
MORENOSÁNCHEZ RAFAEL,
SAAVEDRA EMMA
Publication year - 2008
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.2008.00359.x
Subject(s) - euglena gracilis , biology , alcohol dehydrogenase , biochemistry , cofactor , nad+ kinase , enzyme , dehydrogenase , amino acid , peptide sequence , euglena , molecular cloning , microbiology and biotechnology , gene , chloroplast
.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 ( Eg ADH). 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 Eg ADH reported here may help to elucidate the roles that different ADHs have on the metabolism of short‐ and long‐chain alcohols in this microorganism.