Substrate characterization of a NAD-dependent secondary alcohol dehydrogenase from Rhodococcus sp. GK1 (CIP 105335)

A NAD-dependent secondary alcohol dehydrogenase (SAD) has been extracted from cells of the sterol-degrading bacterium, Rhodococcus sp. GK1 (CIP 105335). The dehydrogenase was partially purified by means of ammonium sulfate fractionation (60% saturation) and filtration on a Sepharose CL-6B column. The obtained enzyme sample was active with aliphatic secondary alcohols, such as 2-hexanol, and as reductase with aliphatic monoketones and diketones, such as 2-hexanone and 2,3-hexanedione. A hydrophobic environment was required for catalysis: methyl on one side and either methyl, ethyl, propyl, butyl, pentyl or hexyl on the other side of the function being transformed. The Km value for NAD or NADH with, respectively 2-propanol or acetone was around 1.60×10−4 M at pH 7.0 and 30 °C. The enzyme affinity (1/Km) for the examined 2-alcohols and 2-ketones (three to eight C atoms) increased with increasing the chain length. Its activity with 2-octanone was somewhat higher than that with 3-octanone, reflecting a better enzyme affinity for a function positioned at C-2. The Km values for the 2-alcohols (pH 7.0, 30 °C) ranged from 6.0×10−2 M for 2-propanol to 1.8×10−3 M for 2-octanol. Reciprocally, the Km values for the 2-ketones ranged from 6.5×10−2 M for acetone to 2.1×10−3 M for 2-octanone. With 2-hexanol as the substrate, the optimal temperature was around 55 °C and the activation energy of the system was 9.49 kcal/mol. The SAD was specific for the (S)-(+)-stereoisomers of 2-butanol.