Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica:: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism
Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica:: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism
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DOI：
10.1046/j.1365-2958.1998.00826.x
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发表时间：
1998-05-01
影响因子：
3.6
通讯作者：
Heider, J
中科院分区：
文献类型：
--
作者：
Leuthner, B;Leutwein, C;Heider, J
甲苯被反硝化细菌Thauera aromatica缺氧降解为二氧化碳。在该途径中的初始反应是富马酸与甲苯的甲基加成，生成丁二酸苄酯作为第一中间体。我们对催化该反应的酶--丁二酸苄酯合成酶(EC 4.1.99-)进行了纯化，并对其性质进行了研究。该酶对氧高度敏感，含有氧化还原活性的黄素辅因子，但不含铁中心。天然相对分子质量为220 kDa；在十二烷基硫酸钠(SDS)凝胶上检测到94(α)、90(α‘)、12(β)和10 kDa(γ)四个亚基。α和α‘-亚基的N-末端序列相同，表明有一半的α-亚基在C-末端降解而得到α’-亚基。因此，天然酶的组成似乎是α(2)β(2)伽马(2)。克隆了含有琥珀酸苄酯合成酶3个亚基基因的5kb DNA片段。预测的基因产物的质量与电喷雾质谱学测定的亚基的质量完全相关。对衍生的氨基酸序列的分析表明，该酶的大亚基与甘氨酰自由基酶有同源性，特别是在预测的自由基位点附近。与丙酮酸甲酸裂解酶及相关蛋白的相似性最高。丁二酸苄酯合成酶的含有自由基的亚基在甘氨酸基的位置被氧化裂解，产生α‘-亚基。用电喷雾质谱对预测的切割位置进行了验证。此外，还发现了一个编码催化甘氨酰自由基形成的激活蛋白的基因。苄基琥珀酸合成酶和活化酶的四个基因被组织成一个操纵子；它们的转录是由甲苯诱导的。预测基因产物的合成是在大肠杆菌中通过T7启动子/聚合酶系统实现的。
Toluene is anoxically degraded to CO2 by the denitrifying bacterium Thauera aromatica. The initial reaction in this pathway is the addition of fumarate to the methyl group of toluene, yielding benzylsuccinate as the first intermediate. We purified the enzyme catalysing this reaction, benzylsuccinate synthase (EC 4.1.99-), and studied its properties. The enzyme was highly oxygen sensitive and contained a redox-active flavin cofactor, but no iron centres. The native molecular mass was 220 kDa; four subunits of 94 (alpha), 90 (alpha'), 12 (beta) and 10 kDa (gamma) were detected on sodium dodecyl sulphate (SDS) gels. The N-terminal sequences of the alpha- and alpha'-subunits were identical, suggesting a C-terminal degradation of half of the alpha-subunits to give the alpha'-subunit. The composition of native enzyme therefore appears to be alpha(2) beta(2) gamma(2). A 5 kb Segment of DNA containing the genes for the three subunits of benzylsuccinate synthase was cloned and sequenced. The masses of the predicted gene products correlated exactly with those of the subunits, as determined by electrospray mass spectrometry. Analysis of the derived amino acid sequences revealed that the large subunit of the enzyme shares homology to glycyl radical enzymes, particularly near the predicted radical site. The highest similarity was observed with pyruvate formate lyases and related proteins. The radical-containing subunit of benzylsuccinate synthase is oxygenolytically cleaved at the site of the glycyl radical, producing the alpha'-subunit. The predicted cleavage site was verified using electrospray mass spectrometry. In addition, a gene coding for an activating protein catalysing glycyl radical formation was found. The four genes for benzylsuccinate synthase and the activating enzyme are organized as a single operon; their transcription is induced by toluene. Synthesis of the predicted gene products was achieved in Escherichia coli in a T7-promotor/polymerase system.