Purification of a glutathione S-transferase and a glutathione conjugate-specific dehydrogenase involved in isoprene metabolism in Rhodococcus sp. strain AD45

A glutathione S transferase (GST) with activity toward 1,2-eposy-2-methyl-3 -butene (isoprene monoxide) and cis-1,2-dichloroepoxyethane was purified fr om the isoprene-utilizing bacterium Rhodococcus sp. strain AD45, The homodi meric enzyme (two subunits of 27 kDa each) catalyzed the glutathione (GSH)- dependent ring opening of various epoxides, At 5 mM GSH, the enzyme followe d Michaelis-Menten kinetics for isoprene monoxide and cis 1,2-dichloroepoxy ethane, with V-max values of 66 and 2.4 mu mol min(-1) mg of protein(-1) an d K-m values of 0.3 and 0.1 mM for isoprene monoside and cis-1,2-dichloroep oxyethane, respectively, Activities increased linearly with the GSH concent ration up to 25 mM. H-1 nuclear magnetic resonance spectroscopy showed that the product of GSH conjugation to isoprene monoxide was 1-hydroxy-2-glutat hionyl-2-methyl-3-butene (HGMB), Thus, nucleophilic attack of GSH occurred on the tertiary carbon atom of the epoxide ring. HGMB was further converted by an NAD(+)-dependent dehydrogenase, and this enzyme was also purified fr om isoprene-grown cells. The homodimeric enzyme (two subunits of 25 kDa eac h) showed a high activity for HGMB, whereas simple primary and secondary al cohols were not oxidized. The enzyme catalyzed the sequential oxidation of the alcohol function to the corresponding aldehyde and carboxylic acid and followed Michaelis-Menten kinetics,vith respect to NAD(+) and HGMB. The res ults suggest that the initial steps in isoprene metabolism are a monooxygen ase-catalyzed conversion to isoprene monoxide, a GST-catalyzed conjugation to HGMB, and a dehydrogenase-catalyzed two-step oxidation to 2-glutathionyl -2-methyl-3-butenoic acid.