A revised biosynthetic pathway for the cofactor F₄₂₀ in prokaryotes

Area of Science:

• Biochemistry
• Microbiology
• Enzymology

Background:

• Cofactor F420 is essential for redox reactions in bacteria and archaea, impacting processes like antibiotic biosynthesis and methanogenesis.
• The complete biosynthetic pathway and specific enzyme functions for F420 production remain largely unknown.
• Key knowledge gaps include the enzyme for 2-phospho-L-lactate generation and the role of FbiB's FMN-binding domain.

Purpose of the Study:

• To elucidate the F420 biosynthetic pathway by identifying novel enzymes and intermediates.
• To determine the substrate specificity and function of FbiD and FbiB in F420 synthesis.
• To enable heterologous production of F420 in a model organism.

Main Methods:

• X-ray crystallography was used to determine the structure of the guanylyltransferase FbiD.
• Enzymatic assays were performed to identify substrates and intermediates in the F420 pathway.
• A recombinant F420 biosynthetic pathway was constructed and expressed in Escherichia coli.

Main Results:

• The study identified phosphoenolpyruvate, not 2-phospho-L-lactate, as the substrate for FbiD and CofC, forming dehydro-F420-0.
• The FMN-binding C-terminal domain of FbiB was shown to reduce dehydro-F420-0 using FMNH2.
• Mature F420 species were produced when FbiB's reductase activity was combined with its N-terminal ligase activity.
• Successful heterologous production of F420 was achieved in E. coli.

Conclusions:

• The F420 biosynthetic pathway involves FbiD, CofC, and FbiB, with specific substrate and intermediate roles elucidated.
• The reductive activity of FbiB's C-terminal domain is critical for generating mature F420.
• This research enables the biotechnological production of F420, opening avenues for its application.