人酸还原酮双加氧酶催化的双化学反应

A. Deshpande, T. Pochapsky, G. Petsko, D. Ringe
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引用次数: 10

摘要

产氧克雷伯菌蛋氨酸回收途径中的酸性还原酮双加氧酶(ARD)是目前已知的唯一一种天然存在的金属酶,它能仅根据结合在活性位点的二价过渡金属离子(Fe2+或Ni2+)的身份在体内催化不同的反应。含铁同工酶通过O2催化底物1,2-二羟基-3-酮-5-(硫甲基)戊-1-烯(酸还原酮)裂解生成甲酸和蛋氨酸的酮酸前体,而含镍同工酶使用相同的底物催化非通路分流生成甲基硫丙酸、一氧化碳和甲酸。这种双重化学反应最近在体外由小家鼠的ARD (MmARD)证实,提供了哺乳动物ARD表现出金属依赖性催化的第一个例子。我们现在表明,人类ARD (HsARD)也具有依赖金属的双重化学能力。表达并纯化重组HsARD,获得具有单一过渡金属离子结合的均相酶。与MmARD一样,Fe2+结合的HsARD表现出最高的活性,并能催化通路上的化学反应,而Ni2+、Co2+或Mn2+则能催化通路外的化学反应。HsARD同工酶的热稳定性与金属离子特性有关,其中Ni2+结合的HsARD最稳定,其次是Co2+和Fe2+, Mn2+结合的HsARD最不稳定。与细菌ARD一样,溶液核磁共振数据表明,HsARD同工酶可以根据金属离子结合而具有显着的结构差异。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Dual chemistry catalyzed by human acireductone dioxygenase
Acireductone dioxygenase (ARD) from the methionine salvage pathway of Klebsiella oxytoca is the only known naturally occurring metalloenzyme that catalyzes different reactions in vivo based solely on the identity of the divalent transition metal ion (Fe2+ or Ni2+) bound in the active site. The iron-containing isozyme catalyzes the cleavage of substrate 1,2-dihydroxy-3-keto-5-(thiomethyl)pent-1-ene (acireductone) by O2 to formate and the ketoacid precursor of methionine, whereas the nickel-containing isozyme uses the same substrates to catalyze an off-pathway shunt to form methylthiopropionate, carbon monoxide and formate. This dual chemistry was recently demonstrated in vitro by ARD from Mus musculus (MmARD), providing the first example of a mammalian ARD exhibiting metal-dependent catalysis. We now show that human ARD (HsARD) is also capable of metal-dependent dual chemistry. Recombinant HsARD was expressed and purified to obtain a homogeneous enzyme with a single transition metal ion bound. As with MmARD, the Fe2+-bound HsARD shows the highest activity and catalyzes on-pathway chemistry, whereas Ni2+, Co2+ or Mn2+ forms catalyze off-pathway chemistry. The thermal stability of the HsARD isozymes is a function of the metal ion identity, with Ni2+-bound HsARD being the most stable followed by Co2+ and Fe2+, and Mn2+-bound HsARD being the least stable. As with the bacterial ARD, solution NMR data suggest that HsARD isozymes can have significant structural differences depending upon the metal ion bound.
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