Redox Coenzyme F420 Biosynthesis in Thermomicrobia Involves Reduction by Stand-Alone Nitroreductase Superfamily Enzymes
Redox Coenzyme F420 Biosynthesis in Thermomicrobia Involves Reduction by Stand-Alone Nitroreductase Superfamily Enzymes
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DOI：
10.1128/aem.00457-20
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发表时间：
2020-06-01
影响因子：
4.4
通讯作者：
Lackner, Gerald
中科院分区：
文献类型：
--
作者：
Braga, Daniel;Hasan, Mahmudul;Lackner, Gerald
辅酶F-420是一种参与古菌和细菌氢化物转移反应的氧化还原辅因子。由于依赖于F-420的酶作为生物催化工具吸引了越来越多的兴趣，F-420的生物合成正在被重新考虑。虽然长期以来人们普遍认为F-420的2-磷酸-L-乳酸(2-PL)部分是由游离的2-PL形成的，但最近的研究表明，磷酸烯醇式丙酮酸被结合到放线杆菌中，并且FbiB蛋白的C-末端结构域是硝基还原酶超家族的成员，它能将脱水的F-420转化为饱和的F-420。然而，在放线杆菌之外，情况仍然不清楚，因为这些生物中缺少FbiB，而且NTR家族的酶高度多样化。在这里，我们通过异源表达和体外检测表明，来自Therommicrobia的独立NTR酶具有脱氢-F-420还原酶活性。代谢组分析和蛋白质组学研究证实了所提出的玫瑰热菌的生物合成途径。这些结果阐明了辅酶F-420在一类革兰氏阴性菌中的生物合成途径，重新定义了NTR超家族的功能亚群，为未来在大肠杆菌中大规模生产F-420提供了一种选择。它参与了抗生素的生物合成、外源物质的降解和不对称酶还原等过程，使F-420与生物技术有很大的相关性。最近，在放线杆菌形成F-420的过程中发现了一个新的生物合成步骤，涉及一个属于多功能硝基还原酶(NTR)超家族的酶结构域，而在革兰氏阴性菌中这一过程仍然是模糊的。在这里，我们表明，类似的生物合成途径存在于热微生物中，尽管关键的生物合成酶显示不同的结构域结构，并且只有遥远的亲缘关系。我们的结果揭示了F-420在革兰氏阴性细菌中的生物合成，并完善了关于NTR酶超家族中序列-功能关系的知识。值得注意的是，这些结果提供了在革兰氏阴性模式生物中生产F-420的另一种途径，并揭示了这一途径的另一个生化方面，有待合成微生物学家探索。
Coenzyme F-420 is a redox cofactor involved in hydride transfer reactions in archaea and bacteria. Since F-420-dependent enzymes are attracting increasing interest as tools in biocatalysis, F-420 biosynthesis is being revisited. While it was commonly accepted for a long time that the 2-phospho-L-lactate (2-PL) moiety of F-420 is formed from free 2-PL, it was recently shown that phosphoenolpyruvate is incorporated in Actinobacteria and that the C-terminal domain of the FbiB protein, a member of the nitroreductase (NTR) superfamily, converts dehydro-F-420 into saturated F-420. Outside the Actinobacteria, however, the situation is still unclear because FbiB is missing in these organisms and enzymes of the NTR family are highly diversified. Here, we show by heterologous expression and in vitro assays that stand-alone NTR enzymes from Thermomicrobia exhibit dehydro-F-420 reductase activity. Metabolome analysis and proteomics studies confirmed the proposed biosynthetic pathway in Thermomicrobium roseum. These results clarify the biosynthetic route of coenzyme F-420 in a class of Gram-negative bacteria, redefine functional subgroups of the NTR superfamily, and offer an alternative for large-scale production of F-420 in Escherichia coli in the future.IMPORTANCE Coenzyme F-420 is a redox cofactor of Archaea and Actinobacteria, as well as some Gram-negative bacteria. Its involvement in processes such as the biosynthesis of antibiotics, the degradation of xenobiotics, and asymmetric enzymatic reductions renders F-420 of great relevance for biotechnology. Recently, a new biosynthetic step during the formation of F-420 in Actinobacteria was discovered, involving an enzyme domain belonging to the versatile nitroreductase (NTR) superfamily, while this process remained blurred in Gram-negative bacteria. Here, we show that a similar biosynthetic route exists in Thermomicrobia, although key biosynthetic enzymes show different domain architectures and are only distantly related. Our results shed light on the biosynthesis of F-420 in Gram-negative bacteria and refine the knowledge about sequence-function relationships within the NTR superfamily of enzymes. Appreciably, these results offer an alternative route to produce F-420 in Gramnegative model organisms and unveil yet another biochemical facet of this pathway to be explored by synthetic microbiologists.