van Hylckama Vlieg J E, Kingma J, Kruizinga W, Janssen D B
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, NL-9747 AG Groningen, The Netherlands.
J Bacteriol. 1999 Apr;181(7):2094-101. doi: 10.1128/JB.181.7.2094-2101.1999.
A glutathione S-transferase (GST) with activity toward 1, 2-epoxy-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 Vmax values of 66 and 2.4 micromol min-1 mg of protein-1 and Km 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.
从利用异戊二烯的红球菌属AD45菌株中纯化出一种对1,2 - 环氧 - 2 - 甲基 - 3 - 丁烯（氧化异戊二烯）和顺式 - 1,2 - 二氯环氧乙烷具有活性的谷胱甘肽S - 转移酶（GST）。这种同二聚体酶（每个亚基为27 kDa，共两个亚基）催化各种环氧化物的谷胱甘肽（GSH）依赖性开环反应。在5 mM GSH条件下，该酶对氧化异戊二烯和顺式 - 1,2 - 二氯环氧乙烷遵循米氏动力学，氧化异戊二烯的Vmax值为66 μmol min⁻¹ mg蛋白质⁻¹，Km值为0.3 mM；顺式 - 1,2 - 二氯环氧乙烷的Vmax值为2.4 μmol min⁻¹ mg蛋白质⁻¹，Km值为0.1 mM。活性随GSH浓度增加至25 mM呈线性增加。¹H核磁共振光谱表明，GSH与氧化异戊二烯结合的产物是1 - 羟基 - 2 - 谷胱甘肽基 - 2 - 甲基 - 3 - 丁烯（HGMB）。因此，GSH的亲核攻击发生在环氧化物环的叔碳原子上。HGMB被一种依赖NAD⁺的脱氢酶进一步转化，该酶也从以异戊二烯生长的细胞中纯化得到。这种同二聚体酶（每个亚基为25 kDa，共两个亚基）对HGMB具有高活性，而简单的伯醇和仲醇不被氧化。该酶催化醇官能团依次氧化为相应的醛和羧酸，并对NAD⁺和HGMB遵循米氏动力学。结果表明，异戊二烯代谢的初始步骤是单加氧酶催化转化为氧化异戊二烯、GST催化与HGMB结合以及脱氢酶催化两步氧化为2 - 谷胱甘肽基 - 2 - 甲基 - 3 - 丁烯酸。
