Deshpande Aditi R, Pochapsky Thomas C, Petsko Gregory A, Ringe Dagmar
Department of Biochemistry, Brandeis University, Waltham, MA 02454, USA.
Department of Chemistry, Brandeis University, Waltham, MA 02454, USA.
Protein Eng Des Sel. 2017 Mar 1;30(3):197-204. doi: 10.1093/protein/gzw078.
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.
来自产酸克雷伯菌甲硫氨酸补救途径的乙二醛还原酶（ARD）是唯一已知的天然金属酶，它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反应。含铁同工酶催化底物1,2-二羟基-3-酮-5-（硫甲基）戊-1-烯（乙二醛还原酮）被O2裂解形成甲酸盐和甲硫氨酸的酮酸前体，而含镍同工酶使用相同的底物催化一条旁路分流反应，形成甲基硫代丙酸酯、一氧化碳和甲酸盐。最近，小家鼠ARD（MmARD）在体外证明了这种双重化学性质，这是哺乳动物ARD表现出金属依赖性催化的首个例子。我们现在表明，人类ARD（HsARD）也能够进行金属依赖性双重化学反应。表达并纯化了重组HsARD，以获得结合了单个过渡金属离子的均质酶。与MmARD一样，结合Fe2+的HsARD显示出最高活性并催化正常途径的化学反应，而结合Ni2+、Co2+或Mn2+的形式催化旁路化学反应。HsARD同工酶的热稳定性是金属离子身份的函数，结合Ni2+的HsARD最稳定，其次是Co2+和Fe2+，结合Mn2+的HsARD最不稳定。与细菌ARD一样，溶液核磁共振数据表明，HsARD同工酶可能会因结合的金属离子而存在显著的结构差异。
