A revised biosynthetic pathway for the cofactor F420 in prokaryotes

Ghader Bashiri,Nobuhiko Tokuriki,Martin Middleditch,Colin Scott,Chris Greening,Colin J Jackson,Sreevalsan Sreebhavan,Brian Palmer,James Antoney,Edward N Baker,Blair Ney,Mihir V Shah,Janine Copp,Stephanie M Stuteley,Ehab N M Jirgis

doi:10.1038/s41467-019-09534-x

Abstract

Cofactor F420 plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent. It mediates a variety of important redox transformations involved in bacterial persistence, antibiotic biosynthesis, pro-drug activation and methanogenesis. However, the biosynthetic pathway for F420 has not been fully elucidated: neither the enzyme that generates the putative intermediate 2-phospho-l-lactate, nor the function of the FMN-binding C-terminal domain of the γ-glutamyl ligase (FbiB) in bacteria are known. Here we present the structure of the guanylyltransferase FbiD and show that, along with its archaeal homolog CofC, it accepts phosphoenolpyruvate, rather than 2-phospho-l-lactate, as the substrate, leading to the formation of the previously uncharacterized intermediate dehydro-F420-0. The C-terminal domain of FbiB then utilizes FMNH2 to reduce dehydro-F420-0, which produces mature F420 species when combined with the γ-glutamyl ligase activity of the N-terminal domain. These new insights have allowed the heterologous production of F420 from a recombinant F420 biosynthetic pathway in Escherichia coli.

Highlights

Cofactor F420 plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent
We found that when Mtb-FbiD and Mtb-FbiA were included in an assay with 2-PL, GTP, and Fo, no product was formed (Fig. 2b)
We have previously shown that full-length FbiB consists of two domains: an N-terminal domain that is homologous to the archaeal γ-glutamyl ligase CofE21,35, and a C-terminal domain of the NTR fold[36] that binds to flavin mononucleotide (FMN) and has no known function, but is essential for extending the poly-γ-glutamate tail[20]

Summary

Introduction

Cofactor F420 plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent. It mediates a variety of important redox transformations involved in bacterial persistence, antibiotic biosynthesis, pro-drug activation and methanogenesis. The C-terminal domain of FbiB utilizes FMNH2 to reduce dehydro-F420-0, which produces mature F420 species when combined with the γ-glutamyl ligase activity of the N-terminal domain These new insights have allowed the heterologous production of F420 from a recombinant F420 biosynthetic pathway in Escherichia coli. It has been hypothesized that a 2-phospho-L-lactate guanylyltransferase (CofC in archaea and the putative enzyme FbiD in bacteria) catalyzes the guanylylation of 2-phospho-L-lactate (2-PL) using guanosine-5ʹtriphosphate (GTP), yielding L-lactyl-2-diphospho-5ʹ-guanosine (LPPG)[17].

Methods

Results

Conclusion