Department of Science, Graduate School of Integrated Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan.
Department of Biological Sciences, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan.
Sci Rep. 2017 Jan 6;7:39991. doi: 10.1038/srep39991.
Pyruvic oxime dioxygenase (POD), a key enzyme in heterotrophic nitrification, was purified from Alcaligenes faecalis, and the molecular and catalytic characteristics were reexamined. POD was purified as the homotetramer of the subunit whose molecular weight was 30,000. The deduced amino acid sequence of POD was homologous with a class II aldolase that has been regarded as the Zn-dependent enzyme catalyzing aldol reactions. The recombinant protein showed weak POD activity, and was activated by reconstitution with Fe. Affinity and catalytic constants were estimated at 470 μM and 4.69 sec, respectively. The POD was inactivated by EDTA to remove bound divalent metal cations. A reconstitution experiment demonstrated that Fe, not Zn, is essential for POD activity and that Mn could partially fulfill the function of Fe. A mutant POD with replacement of His, corresponding to one of three Zn-binding ligands in the class II aldolase, by Asn was purified as a homotetrameric protein but showed no catalytic activities. Those results suggest that the POD is homologous to class II aldolase having non-heme Fe as a catalytic center instead of Zn. A possible mechanism of the POD reaction is discussed on the basis of that of a known Fe-dependent dioxygenase.
丙酮肟双加氧酶（POD）是异养硝化作用中的关键酶，本研究从粪产碱杆菌中纯化得到了 POD，并重新研究了其分子和催化特性。POD 以分子量为 30000 的亚基四聚体的形式被纯化。POD 的推导氨基酸序列与被认为是催化醛醇缩合反应的 Zn 依赖性酶的 II 类醛缩酶同源。重组蛋白表现出较弱的 POD 活性，并且通过与 Fe 重组而被激活。亲和力和催化常数分别估计为 470μM 和 4.69sec。EDTA 可使 POD 失活，以去除结合的二价金属阳离子。重组实验表明，Fe 而非 Zn 对于 POD 活性是必需的，Mn 可以部分替代 Fe 的功能。一个突变的 POD，其 II 类醛缩酶中三个 Zn 结合配体之一的 His 被 Asn 取代，被纯化成为四聚体蛋白，但没有催化活性。这些结果表明，POD 与具有非血红素 Fe 作为催化中心而不是 Zn 的 II 类醛缩酶同源。基于已知的 Fe 依赖性加氧酶，讨论了 POD 反应的可能机制。
