Arnould S, Takahashi M, Camadro J M
Laboratoire de Biochimie des Porphyrines, Département de Microbiologie, Institut Jacques-Monod, Paris, France.
Biochemistry. 1998 Sep 15;37(37):12818-28. doi: 10.1021/bi980713i.
Protoporphyrinogen oxidase catalyzes the oxygen-dependent aromatization of protoporphyrinogen IX to protoporphyrin IX and is the molecular target of diphenyl ether-type herbicides. Structural features of yeast protoporphyrinogen oxidase were assessed by circular dichroism studies on the enzyme purified from E. coli cells engineered to overproduce the protein. Coexpression of the bacterial gene ArgU that encodes tRNAAGA,AGG and a low induction temperature for protein synthesis were critical for producing protoporphyrinogen oxidase as a native, active, membrane-bound flavoprotein. The secondary structure of the protoporphyrinogen oxidase was 40.0 +/- 1. 5% alpha helix, 23.5 +/- 2.5% beta sheet, 18.0 +/- 2.0% beta turn, and 18.5 +/- 2.5% random-coil. Purified protoporphyrinogen oxidase appeared to be a monomeric protein that was relatively heat-labile (Tm of 44 +/- 0.5 degreesC). Acifluorfen, a potent inhibitor that competes with the tetrapyrrole substrate, and to a lower extent FAD, the cofactor of the enzyme, protected the protein from thermal denaturation, raising the Tm to 50.5 +/- 0.5 degreesC (acifluorfen) and 46.5 +/- 0.5 degreesC (FAD). However, diphenyleneiodonium, a slow tight-binding inhibitor that competes with dioxygen, did not protect the enzyme from heat denaturation. Acifluorfen binding to the protein increased the activation energy for the denaturation from 15 to 80 kJ.mol-1. The unfolding of the protein was a two-step process, with an initial fast reversible unfolding of the native protein followed by slow aggregation of the unfolded monomers. Functional analysis indicated that heat denaturation caused a loss of enzyme activity and of the specific binding of radiolabeled inhibitor. Both processes occurred in a biphasic manner, with a transition temperature of 45 degreesC.
原卟啉原氧化酶催化原卟啉原IX依赖氧气的芳构化反应生成原卟啉IX，它是二苯醚类除草剂的分子作用靶点。通过对从经过工程改造以过量表达该蛋白的大肠杆菌细胞中纯化得到的酶进行圆二色性研究，评估了酵母原卟啉原氧化酶的结构特征。编码tRNAAGA,AGG的细菌基因ArgU的共表达以及较低的蛋白质合成诱导温度对于产生天然、有活性的膜结合黄素蛋白形式的原卟啉原氧化酶至关重要。原卟啉原氧化酶的二级结构为40.0±1.5%的α螺旋、23.5±2.5%的β折叠、18.0±2.0%的β转角和18.5±2.5%的无规卷曲。纯化后的原卟啉原氧化酶似乎是一种单体蛋白，相对热不稳定（熔点为44±0.5℃）。作为强效抑制剂的三氟羧草醚与四吡咯底物竞争，在较低程度上与该酶的辅因子FAD竞争，可保护该蛋白免受热变性影响，将熔点提高到50.5±0.5℃（三氟羧草醚）和46.5±0.5℃（FAD）。然而，作为与氧气竞争的缓慢紧密结合抑制剂的二亚苯基碘鎓并不能保护该酶免受热变性影响。三氟羧草醚与该蛋白的结合将变性的活化能从15kJ·mol-1提高到80kJ·mol-1。该蛋白的去折叠是一个两步过程，首先是天然蛋白的初始快速可逆去折叠，随后是去折叠单体的缓慢聚集。功能分析表明，热变性导致酶活性丧失以及放射性标记抑制剂的特异性结合丧失。这两个过程均以双相方式发生，转变温度为45℃。
