The underlying molecular mechanisms of flavin-dependent amine oxidases remain relatively poorly understood, even though many of these enzymes have been reported. The nicotine oxidoreductase NicA2 is a crucial enzyme for the first step of nicotine degradation in Pseudomonas putida S16 (DSM 28022). Here, we present the crystal structure of a ternary complex comprising NicA2 residues 21 to 482, flavin adenine dinucleotide (FAD), and nicotine at 2.25 Å resolution. Unlike other, related structures, NicA2 does not have an associated diacyl glycerophospholipid, wraps its substrate more tightly, and has an intriguing exit passage in which nine bulky amino acid residues occlude the release of its toxic product, pseudooxynicotine (PN). The replacement of these bulky residues by amino acids with small side chains effectively increases the catalytic turnover rate of NicA2. Our results indicate that the passage in wild-type NicA2 effectively controls the rate of PN release and thus prevents its rapid intracellular accumulation. It gives ample time for PN to be converted to less-harmful substances by downstream enzymes such as pseudooxynicotine amine oxidase (Pnao) before its accumulation causes cell damage or even death. The temporal metabolic regulation mode revealed in this study may shed light on the production of cytotoxic compounds.

中文翻译：

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分子减速度调节有毒物质的释放，以防止恶臭假单胞菌S16（DSM 28022）对细胞的损害。

尽管已报道了其中许多酶，但对黄素依赖性胺氧化酶的潜在分子机制仍知之甚少。尼古丁氧化还原酶NicA2是恶臭假单胞菌尼古丁降解第一步的关键酶S16（DSM 28022）。在这里，我们介绍了一种三元复合物的晶体结构，该复合物包含NicA2残基21至482，黄素腺嘌呤二核苷酸（FAD）和尼古丁，分辨率为2.25Å。与其他相关结构不同，NicA2没有相关的二酰基甘油磷脂，更紧密地包裹其底物，并且具有一个引人入胜的出口通道，其中九个庞大的氨基酸残基阻碍了其有毒产物假氧烟碱（PN）的释放。用小侧链的氨基酸取代这些庞大的残基可有效提高NicA2的催化转化率。我们的结果表明，在野生型NicA2中传代可有效控制PN释放的速率，从而阻止其快速的细胞内积累。它为PN提供了充足的时间，使其在下游积累的伪氧化烟碱胺氧化酶（Pnao）转化为危害较小的物质之前积累，从而导致细胞受损甚至死亡。在这项研究中揭示的时间代谢调控模式可能会阐明细胞毒性化合物的产生。