Alcohol dehydrogenases (ADHs; E.C. 126.96.36.199) are widely distributed enzymes found in many microorganisms. ADHs are oxidoreductases that catalyze the NAD(P)+-dependent conversion of alcohols to aldehydes or ketones as well as the reverse reaction. The ADH cloned from Rigidoporus vinctus (RvADH) was 1035 bp that encodes a protein of 344 amino acid residues with calculated molecular mass of 38.39 kDa. This ADH is belonging to the medium-chain family (medium-chain dehydrogenase/reductase (MDR) and has the highly conserved GXXGXXG sequence found in the MDR family which found as the coenzyme-binding pocket. To characterize the ADH protein, the coding region was subcloned into an expression vector pET-20b(+) and transformed into E. coli Rosetta (DE3). The recombinant His6-tagged ADH was overexpressed and purified by Ni2+-nitrilotriacetic acid Sepharose. The purified enzyme showed one band on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Michaelis constant (KM) value of the recombinant enzyme for ethanol was 0.79 mM. In substrates specificity analysis showed that RvADH had great oxidative activity toward primary alcohols. However, the less activtiy toward secondary alcohols and alcohol derivatives were compared with ethanol. Regarding the reductase activity showed low or even no activity to aldehydes and ketone. A 3-D structural model of RvADH was modeled based on the known X-ray structure of PaOR (Pseudomonas aeruginosa) via the SWISS-MODEL program and was superimposed to obtain a better structure via the SPDBV-4 program. Superimposition of RvADH (yellow) and PaOR (blue) was shown using protein solid ribbons. Red stars denote the 3 Gly residues as the NAD+ binding site in the RvADH protein.
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