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首页> 外文期刊>Biochemistry >Metal-catalyzed oxidation and mutagenesis studies on the iron(II) binding site of 1-aminocyclopropane-1-carboxylate oxidase
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Metal-catalyzed oxidation and mutagenesis studies on the iron(II) binding site of 1-aminocyclopropane-1-carboxylate oxidase

机译：金属催化的1-氨基环丙烷-1-羧酸氧化酶的铁（II）结合位点的氧化和诱变研究

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摘要

The final step in the biosynthesis of the plant signaling molecule ethylene is catalyzed by 1-aminocyclopropane-1-carboxylate (ACC) oxidase, a member of the non-heme iron(II) dependent family of oxygenases and oxidases, which has a requirement for ascorbate as a co-substrate and carbon dioxide as an activator. ACC oxidase (tomato) has a particularly short half-life under catalytic conditions undergoing metal-catalyzed oxidative (MCO) fragmentation. Sequence comparisons of ACC oxidases with isopenicillin N synthase (IPNS) and members of the 2-oxoglutarate Fe(II) dependent dioxygenases show an aspartate and two of six ACC oxidase conserved histidine residues are completely conserved throughout this subfamily of Fe(II) dependent oxygenases/oxidases. Previous mutagenesis, spectroscopic, and crystallographic studies on IPNS indicate that the two completely conserved histidine and aspartate residues act as Fe(II) ligands. To investigate the role of the conserved aspartate and histidine residues in ACC oxidase (tomato fruit), they were substituted via site-directed mutagenesis. Modified ACC oxidases produced were H39Q, H56Q, H94Q, H177Q, H177D, H177E, D179E, D179N, H177D&D179E, H211Q, H234Q, H234D, and H234E. Among those histidine mutants replaced by glutamine, H39Q, H56Q, H94Q, and H211Q were catalytically active, indicating these histidines are not essential for catalysis. Mutant enzymes H177D, H177Q, D179N, H177D&D179E, H234Q, H234D, and H234E were catalytically inactive consistent with the assignment of H177, D179, and H234 as iron ligands. Replacement of H177 with glutamate or D179 with glutamate resulted in modified ACC oxidases which still effected the conversion of ACC to ethylene, albeit at a very low level of activity, which was stimulated by bicarbonate. The H177D (inactive), H177E (low activity), D179E (low activity), and H234Q (inactive) modified ACC oxidases all underwent MCO fragmentation, indicating that they can bind iron, dioxygen, ACC, and ascorbate. The results suggest that MCO cleavage results from active site-mediated reactions and imply that, while H177, D179, and H234 are all involved in metal ligation during catalysis, ligation to H234 is not required for fragmentation. It is possible that MCO fragmentation results from reaction of incorrectly folded or "primed" ACC oxidase.
机译：植物信号分子乙烯的生物合成的最后一步是通过1-氨基环丙烷-1-羧酸酯（ACC）氧化酶催化的，该酶是非血红素铁（II）依赖的氧化酶和氧化酶家族的成员，需要抗坏血酸盐作为共底物，二氧化碳作为活化剂。在经历金属催化的氧化（MCO）裂解的催化条件下，ACC氧化酶（番茄）的半衰期特别短。 ACC氧化酶与异青霉素N合酶（IPNS）和2-氧代戊二酸Fe（II）依赖的双加氧酶的序列比较显示天冬氨酸和六个ACC氧化酶保守的组氨酸残基在Fe（II）依赖的加氧酶的这个亚家族中是完全保守的/氧化酶。先前对IPNS的诱变，光谱和晶体学研究表明，两个完全保守的组氨酸和天冬氨酸残基充当Fe（II）配体。为了研究保守的天冬氨酸和组氨酸残基在ACC氧化酶（番茄果实）中的作用，通过定点诱变将它们替换。产生的修饰的ACC氧化酶是H39Q，H56Q，H94Q，H177Q，H177D，H177E，D179E，D179N，H177D＆D179E，H211Q，H234Q，H234D和H234E。在那些被谷氨酰胺替代的组氨酸突变体中，H39Q，H56Q，H94Q和H211Q具有催化活性，表明这些组氨酸对于催化不是必需的。突变酶H177D，H177Q，D179N，H177D＆D179E，H234Q，H234D和H234E具有催化失活作用，这与将H177，D179和H234指定为铁配体一致。用谷氨酸代替H177或用谷氨酸替代D179导致修饰的ACC氧化酶，尽管活性很低，但仍然受到ACC向乙烯的转化的影响，这是由碳酸氢盐刺激的。 H177D（非活性），H177E（低活性），D179E（低活性）和H234Q（非活性）修饰的ACC氧化酶均经历了MCO断裂，表明它们可以结合铁，双氧，ACC和抗坏血酸。结果表明，MCO裂解是由活性位点介导的反应引起的，这意味着尽管H177，D179和H234都在催化过程中参与了金属连接，但片段化不需要与H234连接。 MCO片段化可能是由错误折叠或“引发”的ACC氧化酶反应引起的。

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