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

被引:44
作者
Vlieg, JETV
Kingma, J
Kruizinga, W
Janssen, DB
机构
[1] Univ Groningen, Dept Biochem, Gronigen Biomol Sci & Biotechnol Inst, NL-9747 AG Groningen, Netherlands
[2] Univ Groningen, Dept Chim Organ, NL-9747 AG Groningen, Netherlands
[3] Univ Groningen, Dept Mol Inorgan Chem, NL-9747 AG Groningen, Netherlands
关键词
D O I
10.1128/JB.181.7.2094-2101.1999
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
A glutathione S transferase (GST) with activity toward 1,2-eposy-2-methyl-3-butene (isoprene monoxide) and cis-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, with V-max values of 66 and 2.4 mu mol min(-1) mg of protein(-1) and K-m values of 0.3 and 0.1 mM for isoprene monoside and cis-1,2-dichloroepoxyethane, respectively, Activities increased linearly with the GSH concentration up to 25 mM. H-1 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,vith 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.
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页码:2094 / 2101
页数:8
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