A revised biosynthetic pathway for the cofactor F420 in prokaryotes

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.

Fig. 1

The currently accepted bacterial F₄₂₀ biosynthesis pathway. The pathway is composed of two branches. In the first branch tyrosine is condensed with the flavin biosynthesis intermediate 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione by FbiC/CofGH to produce the riboflavin level chromophore Fo. In the second branch, l-lactic acid is phosphorylated in a guanosine-5ʹ-triphosphate (GTP)-dependent manner by the hypothetical l-lactate kinase CofB to produce 2-phospho-l-lactate (2-PL). This compound is subsequently guanylylated by FbiD/CofC to produce the unstable intermediate l-lactyl-2-diphospho-5ʹ-guanosine (LPPG). The two branches merge as the 2-phospho-l-lactyl moiety of LPPG is transferred to Fo through the action of FbiA/CofD to produce F₄₂₀-0. Mature F₄₂₀ is then produced through glutamylation of F₄₂₀-0 by FbiB/CofE. “Fbi” refers to bacterial proteins, whereas “Cof” represents archaeal ones

Fig. 2

Phosphoenolpyruvate (PEP) is an intermediate in the formation of dehydro-F₄₂₀-0. a Production of F₄₂₀-0 in our revised biosynthesis pathway (left) compared to the currently accepted pathway (right). b Coupled-reaction high-performance liquid chromatography (HPLC) assays showing that both Mtb-FbiD and CofC from Methanocaldococcus jannaschii (Mj-CofC) enzymes use PEP to produce dehydro-F₄₂₀-0. c Tandem mass spectral identification of dehydro-F₄₂₀-0. Tandem mass spectrometry (MS/MS) fragmentation of dehydro-F₄₂₀-0, showing fragment ions with their corresponding structures. The inset displays the observed spectrum of the parent molecule (expected monoisotopic m/z 512.0711 [M − H]⁻)

Fig. 3

Crystal structure of Mtb-FbiD. a Electrostatic surface representation of the Mtb-FbiD structure in complex with phosphoenolpyruvate (PEP), shown as a ball-and-stick model. b The phosphate group of PEP binds to three aspartic acid side chains through two Mg²⁺ ions (shown in cyan). PEP is shown in 2F_o − F_c omit density contoured at 2.0σ, and drawn as ball-and-stick model. Water molecules are shown as red spheres and hydrogen bond interactions are outlined as dashed lines. c Superposition of Mtb-FbiD (wheat ribbon) and Methanocaldococcus jannaschii (Mj-CofC) (green ribbon), indicating 1.85 Å root mean square difference (rmsd) over 181 superimposed Cα. PEP is shown as a ball-and-stick model

Fig. 4

Mtb-FbiB catalyzes reduction of dehydro-F₄₂₀-0. a F₄₂₀-1 is produced in FbiD:FbiA:FbiB coupled assays in the presence of Fre/FMNH₂ and l-glutamate. Tandem mass spectrometry (MS/MS) confirmation of F₄₂₀-1 in both negative (643.12811, [M − H]⁻) and positive (645.27094, [M + H]⁺) modes. b Mtb-FbiB is a bifunctional enzyme catalyzing the reduction of dehydro-F₄₂₀-0 and its poly-glutamylation to form F₄₂₀-n. c Docking of FMNH₂ and dehydro-F₄₂₀-0 into the crystal structure of FbiB C-terminal domain. The methylene group of the enolpyruvyl moiety sits in a pocket made up of M372 and P289, while the carboxylate hydrogen bonds with R337. The methylene double bond sits planar above the isoalloxazine ring of FMNH₂ at an appropriate distance (3.6 Å, shown by dashed line) and oriented for a hydride transfer to the Si face of the methylene bond, accounting for the observed (S)-lactyl moiety of F₄₂₀

Fig. 5

Heterologous expression of F₄₂₀ biosynthesis pathway in E. coli. a Schematic representation of the vector generated for expression of the F₄₂₀ biosynthesis pathway. b High-performance liquid chromatography-fluorescence detector (HPLC-FLD) traces of E. coli lysates containing F₄₂₀ biosynthesis constructs as well as a purified standard from M. smegmatis. c Kinetic studies indicate an identical Michaelis constant for glucose-6-phosphate dehydrogenase (FGD) as measured with F₄₂₀ purified from M. smegmatis (blue squares) and E. coli (red circles). Error bars represent standard deviations. d Fragmentation of F₄₂₀-4 and F₄₂₀-5 extracted from E. coli shows a mature F₄₂₀ production

Fig. 6

The revised bacterial F₄₂₀ biosynthesis pathway. The revised pathway is a modified scheme showing that phosphoenolpyruvate (PEP) acts as the substrate for the FbiD/CofC enzymes to produce enolpyruvyl-diphospho-5ʹ-guanosine (EPPG) or enolpyruvyl-diphospho-5ʹ-adenosine (EPPA) (in the case of CofC). The immediate reaction product formed from Fo and EPPG/EPPA is dehydro-F₄₂₀-0, which is reduced to F₄₂₀-0 through the newly described reductase activity of the C-terminal domain of FbiB in mycobacteria. A separate enzyme in archaea and some bacteria is expected to catalyze this reduction step (CofX). FbiB/CofE subsequently adds a poly-γ-glutamate tail to form F₄₂₀. “Fbi” refers to bacterial proteins, whereas “Cof” represents archaeal ones