期刊
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
卷 86, 期 12, 页码 -出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/AEM.00457-20
关键词
coenzyme; biosynthesis; nitroreductase
资金
- Carl Zeiss Foundation
- Deutsche Forschungsgemeinschaft (DFG) [408113938]
- Leibniz Association
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.
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