1-Aminocyclopropane carboxylic acid oxidase (ACCO) catalyzes the last step of ethylene biosynthesis in plants. Although some sets of structures have been described, there are remaining questions on the active conformation of ACCO and in particular, on the conformation and potential flexibility of the C-terminal part of the enzyme. Several techniques based on the introduction of a probe through chemical modification of amino acid residues have been developed for determining the conformation and dynamics of proteins. Cysteine residues are recognized as convenient targets for selective chemical modification of proteins, thanks to their relatively low abundance in protein sequences and to their well-mastered chemical reactivity. ACCOs have generally 3 or 4 cysteine residues in their sequences. By a combination of approaches including directed mutagenesis, activity screening on cell extracts, biophysical and biochemical characterization of purified enzymes, we evaluated the effect of native cysteine replacement and that of insertion of cysteines on the C-terminal part in tomato ACCO. Moreover, we have chosen to use paramagnetic labels targeting cysteine residues to monitor potential conformational changes by electron paramagnetic resonance (EPR). Given the level of conservation of the cysteines in ACCO from different plants, this work provides an essential basis for the use of cysteine as probe-anchoring residues.

中文翻译：

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1-氨基环丙烷羧酸（ACC）氧化酶的化学修饰：半胱氨酸突变分析，表征和带有氮氧化物自旋标记的生物缀合。

1-氨基环丙烷羧酸氧化酶（ACCO）催化植物中乙烯生物合成的最后一步。尽管已经描述了一些结构集，但是仍然存在关于ACCO的活性构象，特别是关于酶的C末端部分的构象和潜在的柔性的问题。已经开发了几种通过对氨基酸残基进行化学修饰而引入探针的技术，用于确定蛋白质的构象和动力学。半胱氨酸残基被认为是蛋白质选择性化学修饰的方便靶标，这是由于半胱氨酸残基在蛋白质序列中相对较低的丰度以及良好的化学反应性。ACCO通常在其序列中具有3或4个半胱氨酸残基。通过包括定向诱变，对细胞提取物的活性筛选，纯化的酶的生物物理和生化特性在内的多种方法的组合，我们评估了天然半胱氨酸替代和半胱氨酸插入番茄ACCO C端部分的影响。此外，我们选择使用靶向半胱氨酸残基的顺磁性标记物通过电子顺磁共振（EPR）监测潜在的构象变化。鉴于不同植物中ACCO中半胱氨酸的保守程度，这项工作为使用半胱氨酸作为探针固定残基提供了必要的基础。我们评估了天然半胱氨酸替代和半胱氨酸插入对番茄ACCO C末端部分的影响。此外，我们选择使用靶向半胱氨酸残基的顺磁性标记物通过电子顺磁共振（EPR）监测潜在的构象变化。鉴于不同植物中ACCO中半胱氨酸的保守程度，这项工作为使用半胱氨酸作为探针固定残基提供了必要的基础。我们评估了天然半胱氨酸替代和半胱氨酸插入对番茄ACCO C末端部分的影响。此外，我们选择使用靶向半胱氨酸残基的顺磁性标记物通过电子顺磁共振（EPR）监测潜在的构象变化。鉴于不同植物中ACCO中半胱氨酸的保守水平，这项工作为使用半胱氨酸作为探针固定残基提供了必要的基础。