A glutathione-dependent formaldehyde-activating enzyme (Gfa) from Paracoccus denitrificans detected and purified via two-dimensional proton exchange NMR spectroscopy
A glutathione-dependent formaldehyde-activating enzyme (Gfa) from Paracoccus denitrificans detected and purified via two-dimensional proton exchange NMR spectroscopy
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DOI：
10.1074/jbc.c100579200
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发表时间：
2002-02-01
影响因子：
4.8
通讯作者：
Vorholt, JA
中科院分区：
文献类型：
--
作者：
Goenrich, M;Bartoschek, S;Vorholt, JA
由甲醛和谷胱甘肽形成 S-羟甲基谷胱甘肽是一些甲基营养细菌以及许多其他生物体消耗细胞毒素甲醛的核心反应。我们在这里描述了从脱氮副球菌中发现的一种酶，它可以加速这种自发缩合反应。 S-羟甲基谷胱甘肽形成和裂解的速率通过二维质子交换核磁共振波谱在平衡条件下测定。根据反应的温度依赖性和非催化反应的信噪比估计伪一级速率常数k(1)*。在 303 K 和 pH 6.0 k(1)* 条件下，自发反应的时间为 0.02 s(-1)。添加在甲醇存在下生长的脱氮假单胞菌的细胞提取物后，观察到速率常数增加了 10 倍，对应于 35 单位 mg(-1) 的比活性。在琥珀酸盐存在下生长的细胞提取物显示出较低的比活性，为 11 单位 mg(-1)。催化甲醛和谷胱甘肽转化的酶被纯化并命名为谷胱甘肽依赖性甲醛激活酶（Gfa）。基因 gfa 位于谷胱甘肽依赖性甲醛脱氢酶基因的直接上游，该酶催化 S-羟甲基谷胱甘肽的后续氧化。与来自脱氮假单胞菌的 Gfa 具有序列同一性的推定蛋白质也存在于球形红杆菌、苜蓿中华根瘤菌和洛地中生根瘤菌中。
The formation of S-hydroxymethylglutathione from formaldehyde and glutathione is a central reaction in the consumption of the cytotoxin formaldehyde in some methylotrophic bacteria as well as in many other organisms. We describe here the discovery of an enzyme from Paracoccus denitrificans that accelerates this spontaneous condensation reaction. The rates of S-hydroxymethylglutathione formation and cleavage were determined under equilibrium conditions via two-dimensional proton exchange NMR spectroscopy. The pseudo first order rate constants k(1)* were estimated from the temperature dependence of the reaction and the signal to noise ratio of the uncatalyzed reaction. At 303 K and pH 6.0 k(1)* was found to be 0.02 s(-1) for the spontaneous reaction. A 10-fold increase of the rate constant was observed upon addition of cell extract from P. denitrificans grown in the presence of methanol corresponding to a specific activity of 35 units mg(-1). Extracts of cells grown in the presence of succinate revealed a lower specific activity of 11 units mg(-1). The enzyme catalyzing the conversion of formaldehyde and glutathione was purified and named glutathione-dependent formaldehyde-activating enzyme (Gfa). The gene gfa is located directly upstream of the gene for glutathione-dependent formaldehyde dehydrogenase, which catalyzes the subsequent oxidation of S-hydroxymethylglutathione. Putative proteins with sequence identity to Gfa from P. denitrificans are present also in Rhodobacter sphaeroides, Sinorhizobium meliloti, and Mesorhizobium loti.