Dataset Information

Redox Coenzyme F₄₂₀ Biosynthesis in Thermomicrobia Involves Reduction by Stand-Alone Nitroreductase Superfamily Enzymes.

ABSTRACT: 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 a long time that the 2-phospho-l-lactate (2-PL) moiety of F₄₂₀ 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-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 stand-alone NTR enzymes from Thermomicrobia exhibit dehydro-F₄₂₀ reductase activity. Metabolome analysis and proteomics studies confirmed the proposed biosynthetic pathway in Thermomicrobium roseum These results clarify the biosynthetic route of coenzyme F₄₂₀ in a class of Gram-negative bacteria, redefine functional subgroups of the NTR superfamily, and offer an alternative for large-scale production of F₄₂₀ in Escherichia coli in the future.IMPORTANCE Coenzyme F₄₂₀ 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 F₄₂₀ of great relevance for biotechnology. Recently, a new biosynthetic step during the formation of F₄₂₀ 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 F₄₂₀ 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 F₄₂₀ in Gram-negative model organisms and unveil yet another biochemical facet of this pathway to be explored by synthetic microbiologists.

SUBMITTER: Braga D

PROVIDER: S-EPMC7267189 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Redox Coenzyme F420 Biosynthesis in Thermomicrobia Involves Reduction by Stand-Alone Nitroreductase Superfamily Enzymes.

Braga Daniel D Hasan Mahmudul M Kröber Tabea T Last Daniel D Lackner Gerald G

Applied and environmental microbiology 20200602 12

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 a long time that the 2-phospho-l-lactate (2-PL) moiety of F420 is formed from free 2-PL, it was recently shown that phosphoenolpyruvate is incorporated in Actinobacteria and that the C-ter ...[more]

PMID: 32276981

Similar Datasets

Project description:Coenzyme F420 is a microbial redox cofactor that mediates diverse physiological functions and is increasingly used for biocatalytic applications. Recently, diversified biosynthetic routes to F420 and the discovery of a derivative, 3PG-F420, were reported. 3PG-F420 is formed via activation of 3-phospho-d-glycerate (3-PG) by CofC, but the structural basis of substrate binding, its evolution, as well as the role of CofD in substrate selection remained elusive. Here, we present a crystal structure of the 3-PG-activating CofC from Mycetohabitans sp. B3 and define amino acids governing substrate specificity. Site-directed mutagenesis enabled bidirectional switching of specificity and thereby revealed the short evolutionary trajectory to 3PG-F420 formation. Furthermore, CofC stabilized its product, thus confirming the structure of the unstable molecule and revealing its binding mode. The CofD enzyme was shown to significantly contribute to the selection of related intermediates to control the specificity of the combined biosynthetic CofC/D step. These results imply the need to change the design of combined CofC/D activity assays. Taken together, this work presents novel mechanistic and structural insights into 3PG-F420 biosynthesis and evolution and opens perspectives for the discovery and enhanced biotechnological production of coenzyme F420 derivatives in the future. IMPORTANCE The microbial cofactor F420 is crucial for processes like methanogenesis, antibiotics biosynthesis, drug resistance, and biocatalysis. Recently, a novel derivative of F420 (3PG-F420) was discovered, enabling the production and use of F420 in heterologous hosts. By analyzing the crystal structure of a CofC homolog whose substrate choice leads to formation of 3PG-F420, we defined amino acid residues governing the special substrate selectivity. A diagnostic residue enabled reprogramming of the substrate specificity, thus mimicking the evolution of the novel cofactor derivative. Furthermore, a labile reaction product of CofC was revealed that has not been directly detected so far. CofD was shown to provide another layer of specificity of the combined CofC/D reaction, thus controlling the initial substrate choice of CofC. The latter finding resolves a current debate in the literature about the starting point of F420 biosynthesis in various organisms.

Dataset Information

Redox Coenzyme F₄₂₀ Biosynthesis in Thermomicrobia Involves Reduction by Stand-Alone Nitroreductase Superfamily Enzymes.

Publications

Redox Coenzyme F<sub>420</sub> Biosynthesis in <i>Thermomicrobia</i> Involves Reduction by Stand-Alone Nitroreductase Superfamily Enzymes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets