N-methylation is a recurring feature in the biosynthesis of many plant specialized metabolites, including alkaloids. A crucial step in the conserved central pathway that provides intermediates for the biosynthesis of benzylisoquinoline alkaloids (BIAs) involves conversion of the secondary amine (S)-coclaurine into the tertiary amine (S)-N-methylcoclaurine by coclaurine N-methyltransferase (CNMT). Subsequent enzymatic steps yield the core intermediate (S)-reticuline, from which various branch pathways for the biosynthesis of major BIAs such as morphine, noscapine and sanguinarine diverge. An additional N-methylation yielding quaternary BIAs is catalyzed by reticuline N-methyltransferase (RNMT), such as in the branch pathway leading to the taxonomically widespread and ecologically significant alkaloid magnoflorine. Despite their functional differences, analysis of primary sequence information has been unable to accurately distinguish between CNMT-like and RNMT-like enzymes, necessitating laborious in vitro screening. Furthermore, despite a recent emphasis on structural characterization of BIA NMTs, the features and mechanisms underlying the CNMT-RNMT functional dichotomy were unknown. We report the identification of structural variants tightly correlated with function in known BIA NMTs and show through reciprocal mutagenesis that a single residue acts as a switch between CNMT- and RNMT-like functions. We use yeast in vivo screening to show that this discovery allows for accurate prediction of activity strictly from primary sequence information and, on this basis, improve the annotation of previously reported putative BIA NMTs. Our results highlight the unusually short mutational distance separating ancestral CNMT-like enzymes from more evolutionarily advanced RNMT-like enzymes, and thus help explain the widespread yet sporadic occurrence of quaternary BIAs in plants. While this is the first report of structural variants controlling mono-versus di-methylation activity among plant NMT enzymes, comparison with bacterial MT enzymes also suggests possible convergent evolution.

中文翻译：

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单个残基决定了苄基异喹啉生物碱 N-甲基转移酶的底物偏好

N-甲基化是许多植物特殊代谢物（包括生物碱）生物合成中的重复特征。为苄基异喹啉生物碱 (BIA) 的生物合成提供中间体的保守中央途径中的一个关键步骤涉及通过 coclaurine N-甲基转移酶 (CNMT) 将仲胺 (S)-coclaurine 转化为叔胺 (S)-N-methylcoclaurine . 随后的酶促步骤产生核心中间体 (S)-网状番荔枝碱，主要 BIA（如吗啡、那可品和血根碱）的生物合成的各种分支途径从中分出。产生季铵盐 BIA 的额外 N-甲基化由网状番荔枝碱 N-甲基转移酶 (RNMT) 催化，例如在导致分类学上广泛且生态上重要的生物碱木兰素的分支途径中。尽管它们的功能存在差异，但对一级序列信息的分析无法准确地区分 CNMT 样和 RNMT 样酶，需要费力的体外筛选。此外，尽管最近强调了 BIA NMT 的结构特征，但 CNMT-RNMT 功能二分法的特征和机制尚不清楚。我们报告了与已知 BIA NMT 功能紧密相关的结构变体的鉴定，并通过相互诱变表明单个残基充当 CNMT 和 RNMT 样功能之间的转换。我们使用酵母体内筛选来表明这一发现允许严格根据一级序列信息准确预测活性，并在此基础上改进先前报告的假定 BIA NMT 的注释。我们的结果突出了异常短的突变距离，将祖先的 CNMT 样酶与进化上更先进的 RNMT 样酶分开，从而有助于解释植物中第四纪 BIA 的广泛但零星发生。虽然这是关于控制植物 NMT 酶中单甲基化和二甲基化活性的结构变异的第一份报告，但与细菌 MT 酶的比较也表明可能的趋同进化。