Biosynthesis of the redox coenzyme F420 in Thermomicrobia involves reduction by standalone nitroreductase superfamily enzymes.

Coenzyme F420 is a redox cofactor involved in hydride transfer reactions in archaea and bacteria. Since F420-dependent enzymes are attracting increasing interest as tools in biocatalysis, F420 biosynthesis is being revisited. While it was commonly accepted for long that the 2-phospho-l-lactate (2-PL) moiety of F420 is formed from free 2-PL, it was recently shown that PEP is incorporated in Actinobacteria and that the C-terminal domain of the FbiB protein, a member of the nitroreductase superfamily (NTR), converts dehydro-F420 into saturated F420. 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 standalone NTR enzymes from Thermomicrobia exhibit dehydro-F420 reductase activity. Metabolome analysis and proteomics studies confirmed the proposed biosynthetic pathway in Thermomicrobium roseum. These results clari...