Purification of a Glutathione S -Transferase and a Glutathione Conjugate-Specific Dehydrogenase Involved in Isoprene Metabolism in Rhodococcus sp. Strain AD45

Abstract

A glutathione S-transferase (GST) with activity toward 1,2-epoxy-2-methyl-3-butene (isoprene monoxide) andcis-1,2-dichloroepoxyethane was purified from the isoprene-utilizing bacterium Rhodococcus sp. strain AD45. The homodimeric enzyme (two subunits of 27 kDa each) catalyzed the glutathione (GSH)-dependent ring opening of various epoxides. At 5 mM GSH, the enzyme followed Michaelis-Menten kinetics for isoprene monoxide and cis-1,2-dichloroepoxyethane, withV max values of 66 and 2.4 μmol min−1 mg of protein−1 andKm values of 0.3 and 0.1 mM for isoprene monoxide and cis-1,2-dichloroepoxyethane, respectively. Activities increased linearly with the GSH concentration up to 25 mM.1H nuclear magnetic resonance spectroscopy showed that the product of GSH conjugation to isoprene monoxide was 1-hydroxy-2-glutathionyl-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 from isoprene-grown cells. The homodimeric enzyme (two subunits of 25 kDa each) showed a high activity for HGMB, whereas simple primary and secondary alcohols 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 with respect to NAD+ and HGMB. The results suggest that the initial steps in isoprene metabolism are a monooxygenase-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.

Identification and characterisation of isoprene‐degrading bacteria in an estuarine environment

“…In Rhodococcus sp. AD45, IsoI catalyses the conjugation of isoprene epoxide with glutathione and IsoH is responsible for the two subsequent oxidation reactions, while no known function has been assigned to IsoG or IsoJ (van Hylckama Vlieg et al, 1999). In Rhodococcus sp.…”

“…Isoprene metabolism is best characterised in Rhodococcus sp. AD45 (van Hylckama Vlieg et al, 1998;van Hylckama Vlieg et al, 1999;van Hylckama Vlieg et al, 2000;Crombie et al, 2015), originally isolated from freshwater sediment, which can grow on isoprene as sole carbon and energy source. The genome includes genes for a multicomponent IsoMO essential for isoprene metabolism (Crombie et al, 2015), most similar to other soluble diiron centre monooxygenases (SDIMOs) such as alkene monooxygenase from the propene (propylene) degrader Xanthobacter autotrophicus Py2 (Small and Ensign, 1997).…”

“…The epoxide ring is cleaved by conjugation with glutathione and catalysed by glutathione-S-transferase (IsoI). A dehydrogenase, IsoH, then oxidises the alcohol moiety of 1-hydroxy-2-glutathionyl-2-methyl-3-butene to the carboxylic acid (van Hylckama Vlieg et al, 1999). The genes responsible form part of a cluster of 22, all of which were implicated in isoprene metabolism (Crombie et al, 2015).…”

Mycothiol-dependent proteins in actinomycetes

“…More interestingly, this gene also has 29.1% identity in 151-aa overlap with the gene products of fosfomycin resistance genes that serve as GSTs in the conjugation of GSH to a carbon on the epoxide ring of the fosfomycin. Besides diamide, this mutant is sensitive to CDNB, the model GST substrate, and other alkylating agents, but it is not sensitive to either fosfomycin or methyl glyxoal (van Hylckama Vlieg et al, 1999). MSH transferase assays with the recombinant gene product of Rv0274 using CDNB, fosfomycin, and monochlorobimane, a less reactive bimane than monobromobimane, as substrates were unsuccessful (M. Rawat and G. Newton, unpublished results), contradicting the assumption that it is a MSH transferase.…”

Bacterial sources and sinks of isoprene, a reactive atmospheric hydrocarbon

“…Although this enzyme system has not been puri®ed, all six of the proteins encoded by this gene cluster are homologous to proteins in other characterized multicomponent monooxygenases (van Hylckama Vlieg, 1999). 1,2-Epoxy-2-methyl-3-butene is converted to a glutathione conjugate by a glutathione-S-transferase (GST) encoded by IsoI (van Hylckama Vlieg, 1999;van Hylckama Vlieg et al, 1999). This reaction is a critical one, as it prevents the reactive epoxide from alkylating DNA and proteins.…”