The redox regulation of melatonin synthesis in circadian rhythm

5-羟色胺N-乙酰基转移酶（serotoninN-acetyltransferase，EC.2.3.1.87）是松果体中一种褪黑激素节律产生酶。为了建立褪黑激素节律，AA-NAT活性通过几种信号通路精确调节。在这里，我们展示了AA-NAT活性的新调节，其中分子内二硫键可以作为催化的开关。N-乙基马来酰亚胺（NEM）以乙酰辅酶A保护的方式不可逆地抑制乙酰化剂-AA-NAT活性。氧化谷胱甘肽或溶解氧可逆地抑制AA-NAT的乙酰辅酶A保护的方式。为了鉴定负责抑制的半胱氨酸残基，首先用溶解氧氧化AA-NAT，用NEM处理，用二硫苏糖醇还原，然后用[^<14>C]-NEM标记。Cys 61和Cys 177以乙酰辅酶A保护的方式特异性标记。具有Cys 61至Ala和Cys 177至Ala双取代的AA-NAT（Cys 61 Ala/Cys 177 Ala-AA-NAT）是完全活性的，但不表现出对氧化或NEM的敏感性，而仅具有单取代的AA-NAT保留了这些敏感性的约40%。Cys 61和Cys 177之间的分子内二硫键在氧化时形成，并在还原时裂解。此外，在COS 7细胞中表达的Cys 61 Ala/Cys 177 Ala-AA-NAT对H2 O2诱导的氧化应激相对不敏感，而野生型AA-NAT在相同条件下被强烈抑制。这些结果表明，Cys 61和Cys 177之间的二硫键的形成和切割产生的活性和非活性状态的AA-NAT。这是可能的细胞内氧化还原条件调节AA-NAT活性通过切换通过分子内二硫键。

Serotonin N-acetyltransferase (EC.2.3.1.87)(AA-NAT) is a melatonin rhythm-generating enzyme in pineal glands. To establish a melatonin rhythm, AA-NAT activity is precisely regulated through several signaling pathways. Here we show novel regulation of AA-NAT activity, in which an intramolecular disulfide bond may function as a switch for the catalysis. Recombinant-AA-NAT activity was irreversibly inhibited by N-ethylmaleimide(NEM) in an acetyl CoA-protectable manner. Oxidized glutathione or dissolved oxygen reversibly inhibited AA-NAT in an acetyl CoA-protectable manner. To identify the cysteine residues responsible for the inhibition, AA-NAT was first oxidized with dissolved oxygen, treated with NEM, reduced with dithiothreitol, and then labeled with [^<14>C]-NEM. Cys61 and Cys177 were specifically labeled in an acetyl CoA-protectable manner. The AA-NAT with the Cys61 to Ala and Cys177 to Ala double substitutions(Cys61Ala/Cys177Ala-AA-NAT) was fully active but did not exhibit sensitivity to either oxidation or NEM, while the AA-NATs with only the single substitutions retained about 40 % of these sensitivities. An intramolecular disulfide bond between Cys61 and Cys177 formed upon oxidation and cleaved upon reduction was identified. Furthermore, Cys61Ala/Cys177Ala-AA-NAT expressed in COS7 cells was relatively insensitive to H2O2-evoked oxidative stress, while wild type AA-NAT was strongly inhibited under the same conditions. These results indicate that the formation and cleavage of the disulfide bond between Cys61 and Cys177 produce the active and inactive states of AA-NAT. It is possible that intracellular redox conditions regulate AA-NAT activity through switching via an intramolecular disulfide bridge.