Verfürth Knut, Pierik Antonio J, Leutwein Christina, Zorn Susanne, Heider Johann
Mikrobiologie, Albert-Ludwigs-Universität, Schänzlestrasse 1, Institut für Biologie II, 79104 Freiburg, Germany.
Arch Microbiol. 2004 Feb;181(2):155-62. doi: 10.1007/s00203-003-0642-4. Epub 2003 Dec 20.
The anaerobic degradation pathways of toluene and m-xylene are initiated by addition of a fumarate cosubstrate to the methyl group of the hydrocarbon, yielding (R)-benzylsuccinate and (3-methylbenzyl)succinate, respectively, as first intermediates. These reactions are catalyzed by a novel glycyl-radical enzyme, (R)-benzylsuccinate synthase. Substrate specificities of benzylsuccinate synthases were analyzed in Azoarcus sp. strain T and Thauera aromatica strain K172. The enzyme of Azoarcus sp. strain T converts toluene, but also all xylene and cresol isomers, to the corresponding succinate adducts, whereas the enzyme of T. aromatica is active with toluene and all cresols, but not with any xylene isomer. This corresponds to the capabilities of Azoarcus sp. strain T to grow on either toluene or m-xylene, and of T. aromatica to grow on toluene as sole hydrocarbon substrate. Thus, differences in the substrate spectra of the respective benzylsuccinate synthases of the two strains contribute to utilization of different aromatic hydrocarbons, although growth on different substrates also depends on additional determinants. We also provide direct evidence by electron paramagnetic resonance (EPR) spectroscopy that glycyl radical enzymes corresponding to substrate-induced benzylsuccinate synthases are specifically detectable in anoxically prepared extracts of toluene- or m-xylene-grown cells. The presence of the EPR signals and the determined amount of the radical are consistent with the respective benzylsuccinate synthase activities. The properties of the EPR signals are highly similar to those of the prototype glycyl radical enzyme pyruvate formate lyase, but differ slightly from previously reported parameters for partially purified benzylsuccinate synthase.
甲苯和间二甲苯的厌氧降解途径是通过将富马酸酯共底物添加到烃的甲基上启动的，分别产生（R）-苄基琥珀酸酯和（3-甲基苄基）琥珀酸酯作为首个中间体。这些反应由一种新型的甘氨酰自由基酶，即（R）-苄基琥珀酸合酶催化。在偶氮弧菌属菌株T和嗜芳烃陶厄氏菌菌株K172中分析了苄基琥珀酸合酶的底物特异性。偶氮弧菌属菌株T的酶可将甲苯以及所有二甲苯和甲酚异构体转化为相应的琥珀酸加合物，而嗜芳烃陶厄氏菌的酶对甲苯和所有甲酚有活性，但对任何二甲苯异构体均无活性。这与偶氮弧菌属菌株T能够在甲苯或间二甲苯上生长，以及嗜芳烃陶厄氏菌能够以甲苯作为唯一碳氢化合物底物生长的能力相对应。因此，尽管在不同底物上的生长也取决于其他决定因素，但这两种菌株各自的苄基琥珀酸合酶底物谱的差异有助于利用不同的芳烃。我们还通过电子顺磁共振（EPR）光谱提供了直接证据，即在厌氧制备的甲苯或间二甲苯生长细胞的提取物中可特异性检测到与底物诱导的苄基琥珀酸合酶相对应的甘氨酰自由基酶。EPR信号的存在和测定的自由基量与各自的苄基琥珀酸合酶活性一致。EPR信号的特性与原型甘氨酰自由基酶丙酮酸甲酸裂解酶的特性高度相似，但与先前报道的部分纯化的苄基琥珀酸合酶的参数略有不同。
