Unciuleac M, Boll M
Mikrobiologie, Fakultät für Biologie, Universität Freiburg, Schänzlestrasse 1, D-79104 Freiburg, Germany.
Proc Natl Acad Sci U S A. 2001 Nov 20;98(24):13619-24. doi: 10.1073/pnas.241375598. Epub 2001 Nov 6.
Benzoyl-CoA reductase (BCR) from the bacterium Thauera aromatica catalyzes the two-electron reduction of benzoyl-CoA (BCoA) to a nonaromatic cyclic diene. In a process analogous to enzymatic nitrogen reduction, BCR couples the electron transfer to the aromatic ring to a stoichiometric hydrolysis of 2 ATP/2e(-). Reduced but not oxidized BCR hydrolyzes ATP to ADP. In this work, purified BCR was shown to catalyze an isotope exchange from [(14)C]ADP to [(14)C]ATP, which was approximately 15% of the ATPase activity in the presence of equimolar amounts of ADP and ATP. In accordance, BCR (alpha beta gamma delta-composition) autophosphorylated its gamma-subunit when incubated with [gamma-(32)P]ATP. Formation of the enzyme-phosphate was independent of the redox state, whereas only dithionite-reduced BCR catalyzed a dephosphorylation associated with the ATPase activity. This finding suggests that the ATPase- and autophosphatase-partial activities of BCR exhibit identical redox dependencies. BCoA or the nonphysiological electron-accepting substrate hydroxylamine stimulated the redox-dependent effects; the rates of both the overall ATPase and the autophosphatase activities of reduced BCR were increased 6-fold. In contrast, BCoA and hydroxylamine had no effect on oxidized and phosphorylated BCR. The reactivity of the phosphoamino acid fits best with a phosphoamidate or acylphosphate linkage. The results obtained suggest a mechanism of ATP hydrolysis-driven electron transfer, which differs from that of nitrogenase by the transient formation of a phosphorylated enzyme.
来自嗜芳烃陶厄氏菌的苯甲酰辅酶A还原酶（BCR）催化苯甲酰辅酶A（BCoA）进行双电子还原反应，生成一种非芳香族环状二烯。在一个类似于酶促氮还原的过程中，BCR将电子转移至芳香环的过程与2分子ATP/2个电子的化学计量水解相偶联。还原型而非氧化型的BCR可将ATP水解为ADP。在本研究中，纯化后的BCR被证明能催化同位素从[¹⁴C]ADP交换至[¹⁴C]ATP，在等摩尔量的ADP和ATP存在时，该交换量约为ATP酶活性的15%。相应地，BCR（αβγδ组成）与[γ-³²P]ATP一起孵育时会使其γ亚基发生自磷酸化。酶-磷酸的形成与氧化还原状态无关，而只有连二亚硫酸盐还原型的BCR能催化与ATP酶活性相关的去磷酸化反应。这一发现表明，BCR的ATP酶和自磷酸酶部分活性表现出相同的氧化还原依赖性。BCoA或非生理性电子受体底物羟胺可刺激氧化还原依赖性效应；还原型BCR的总体ATP酶活性和自磷酸酶活性速率均提高了6倍。相比之下，BCoA和羟胺对氧化型和磷酸化的BCR没有影响。磷酸化氨基酸的反应性与氨基磷酸酯或酰基磷酸酯键最为匹配。所得结果提示了一种由ATP水解驱动的电子转移机制，该机制与固氮酶的不同之处在于会短暂形成磷酸化酶。
