An Aminotransferase Is Responsible for the Deamination of the N-Terminal Leucine and Required for Formation of Oxazolone Ring A in Methanobactin of Methylosinus trichosporium OB3b
An Aminotransferase Is Responsible for the Deamination of the N-Terminal Leucine and Required for Formation of Oxazolone Ring A in Methanobactin of Methylosinus trichosporium OB3b
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DOI：
10.1128/aem.02619-16
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发表时间：
2017-01-01
影响因子：
4.4
通讯作者：
Semrau, Jeremy D.
中科院分区：
文献类型：
--
作者：
Gu, Wenyu;Baral, Bipin S.;Semrau, Jeremy D.
甲烷氧化菌的基因表达已被证明受到多种金属的可用性的影响，最明显的是铜调节甲烷单加氧酶的替代形式的表达。一种被称为甲烷菌蛋白的铜结合化合物，或称叶绿素，在甲烷氧化菌对铜的吸收中起着关键作用。Methanobactin是一种核糖体合成的翻译后修饰肽（RiPP），由X-Cys二肽序列结合铜形成，具有两个杂环，每个杂环上都有一个相关的硫酰胺。编码甲烷菌素前体多肽的基因mbnA是一个基因簇的一部分，但其他基因在甲烷菌素生物合成中的作用尚不清楚。为了开始阐明这些基因的功能，我们在trichosporium Methylosinus OB3b中构建了一个mbnABCMN的无标记缺失，然后使用宽宿主范围的克隆载体同源表达mbnABCM，以确定mbnN的功能，注释为编码转氨酶。该菌株产生的甲烟碱与野生型的甲烟碱有很大的不同，缺少c端蛋氨酸，并且只形成两个恶唑酮环中的一个。相反，在M. trichosporium OB3b中n端3-甲基丁烷-恶唑酮-硫酰胺基团的位置，发现了亮氨酸和含硫酰胺的甘氨酸（Gly-psi），这表明MbnN用于甲烷obactin的n端亮氨酸的脱胺，并且这种翻译后修饰对于n端恶唑酮环的关闭至关重要。这些研究为甲烷obactin的生物合成提供了新的见解，也为了解甲烷obactin基因簇中其他基因的功能提供了一个平台。甲烷氧化菌是在碳循环中起关键作用的微生物，受铜的影响，其基因表达和酶活性随着铜水平的变化而变化。甲烷氧化菌产生一种与铜结合的化合物，称为甲烷菌素，用于吸收铜，甲烷菌素在控制甲烷氧化活性中起关键作用。Methanobactin也被证明对Wilson病（一种常染色体隐性遗传病，人体不能正确吸收铜）的治疗有效。表征甲烷菌素的生物合成途径对于了解甲烷氧化菌如何对其环境做出反应以及优化甲烷菌素用于治疗Wilson病等与铜相关的疾病具有重要意义。本研究表明，编码转氨酶的mbnN参与了n端亮氨酸的脱氨作用，并且对于甲烷菌中一个负责铜结合的杂环的形成是必需的，而不是两个杂环。
Gene expression in methanotrophs has been shown to be affected by the availability of a variety of metals, most notably copper-regulating expression of alternative forms of methane monooxygenase. A copper-binding compound, or chalkophore, called methanobactin plays a key role in copper uptake in methanotrophs. Methanobactin is a ribosomally synthesized and posttranslationally modified peptide (RiPP) with two heterocyclic rings with an associated thioamide for each ring, formed from X-Cys dipeptide sequences that bind copper. The gene coding for the precursor polypeptide of methanobactin, mbnA, is part of a gene cluster, but the role of other genes in methanobactin biosynthesis is unclear. To begin to elucidate the function of these genes, we constructed an unmarked deletion of mbnABCMN in Methylosinus trichosporium OB3b and then homologously expressed mbnABCM using a broad-host-range cloning vector to determine the function of mbnN, annotated as coding for an aminotransferase. Methanobactin produced by this strain was found to be substantially different from wild-type methanobactin in that the C-terminal methionine was missing and only one of the two oxazolone rings was formed. Rather, in place of the N-terminal 3-methylbutanoyl-oxazolone-thioamide group, a leucine and a thioamide-containing glycine (Gly-psi) were found, indicating that MbnN is used for deamination of the N-terminal leucine of methanobactin and that this posttranslational modification is critical for closure of the N-terminal oxazolone ring in M. trichosporium OB3b. These studies provide new insights into methanobactin biosynthesis and also provide a platform for understanding the function of other genes in the methanobactin gene cluster.IMPORTANCE Methanotrophs, microbes that play a critical role in the carbon cycle, are influenced by copper, with gene expression and enzyme activity changing as copper levels change. Methanotrophs produce a copper-binding compound, or chalkophore, called methanobactin for copper uptake, and methanobactin plays a key role in controlling methanotrophic activity. Methanobactin has also been shown to be effective in the treatment of Wilson disease, an autosomal recessive disorder where the human body cannot correctly assimilate copper. It is important to characterize the methanobactin biosynthesis pathway to understand how methanotrophs respond to their environment as well as to optimize the use of methanobactin for the treatment of copper-related diseases such as Wilson disease. Here we show that mbnN, encoding an aminotransferase, is involved in the deamination of the N-terminal leucine and necessary for the formation of one but not both of the heterocyclic rings in methanobactin that are responsible for copper binding.