INTRODUCTION
Organic cofactors like thiamin pyrophosphate (TPP), flavins (FAD, FMN), or nicotinamides (NAD
+, NADP
+) are small molecules other than amino acids that are required for the catalytic activity of enzymes (
1). Knowledge about cofactor structure, function, and biosynthesis is therefore crucial to understand the biochemical and physiological processes their dependent enzymes are involved in and facilitates the biotechnological exploitation of these enzymes. Coenzyme F
420 is a specialized redox cofactor that was so far mainly identified in archaea and some actinobacteria (
2). In archaea, F
420 is a key coenzyme of methanogenesis (
3). In mycobacteria, F
420 plays a vital role in respiration (
4,
5), cell wall biosynthesis (
6,
7), as well as the activation of medicinally relevant antimycobacterial (pro-)drugs. For instance, the novel anti-tubercular drug pretomanid is activated by Ddn, an F
420-dependent nitroreductase (
8,
9). In streptomycetes, F
420H
2 is used for reduction steps during the biosynthesis of antibiotics like thiopeptins (
10), lanthipeptides (
11), or oxytetracycline (
12,
13). Increasing interest in F
420 is also driven by the utilization of F
420H
2-dependent reductases in biocatalysis, for example, for asymmetric ene reductions (
14–18).
Intriguingly, F
420 also occurs in a few Gram-negative bacteria where it has been acquired most likely by horizontal transfer of its biosynthetic genes from actinobacteria (
19,
20). Initial studies have revealed that F
420 is indeed produced by some of these organisms but their physiological role remains unknown (
20,
21). We have recently identified F
420 biosynthetic genes in the genome of
Mycetohabitans (synonym:
Paraburkholderia)
rhizoxinica (
22), a symbiont that inhabits the hyphae and spores of the phytopathogenic mold
Rhizopus microsporus (
23–26). Surprisingly, we discovered that the symbiont produced a novel derivative of F
420, which we termed 3PG-F
420 (
22). The cofactor activity of 3PG-F
420 was comparable to classical F
420 and could serve as a substitute for the latter in biocatalysis (
22). Although this congener has not been described in any other organism, it could also be detected in the microbiota of a biogas production plant, thus demonstrating that it is not restricted to endofungal bacteria (
22). The producers of 3PG-F
420 in these habitats, however, are unknown. We hypothesize that analysis of organisms that have evolved a derivative of an otherwise conserved cofactor may also harbor unusual enzyme families that utilize this cofactor derivative. These enzymes could have novel activities or substrate specificities and are therefore of potential interest for biocatalysis.
The biosynthesis of 3PG-F
420 (
Fig. 1) is generally similar to the biosynthesis of classical F
420 (
27). The pathway starts with the formation of the redox-active core moiety 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F
O) from
l-tyrosine and 5-amino-6-ribitylamino-uracil, a reactive metabolite of the flavin biosynthesis pathway. The F
O core is then elongated by a chemical group that can formally be described as 2-phospho-
l-lactate (2-PL) before an oligoglutamate tail is added. The biosynthesis of the 2-PL moiety has been the subject of several studies. Seminal work on archaea suggested that it is directly formed from 2-phospho-
l-lactate: Incubation of cell extracts of
Methanosarcina thermophila or
Methanocaldococcus jannaschii with F
O, 2-PL, and GTP led to the formation of F
420-0 (
28). Biochemical assays with purified CofC and CofD finally corroborated the model that the guanylyltransferase CofC catalyzes the reaction of 2-PL and GTP to lactyl-2-phospho-guanosine (LPPG) (
29), which is then passed on to CofD to transfer the activated 2-PL moiety onto the precursor F
O. However, the unstable nature of LPPG has prevented confirmation of its structure by NMR or mass spectrometry so far. The last biosynthetic step leading to the mature coenzyme F
420 is catalyzed by the F
420:glutamyl ligase CofE (
30), which is responsible for the addition of the
γ-linked oligoglutamate moiety to the F
420-0 core, thus forming F
420-n, with n indicating the number of glutamate residues.
In mycobacteria, CofE is not a free-standing enzyme but constitutes the
N-terminal domain of the FbiB protein (
31). It was shown recently that mycobacteria utilize phosphoenolpyruvate (PEP), but not 2-PL, to form F
420-0. Instead of LPPG, EPPG is formed, which is converted into dehydro-F
420-0 (DF
420-0) by the action of FbiA, the mycobacterial CofD homolog. DF
420-0 is then reduced to classical F
420-0 by the
C-terminal domain of FbiB, which belongs to the nitroreductase superfamily (
32). We have shown that a similar pathway is present in the thermophilic bacterium
Thermomicrobium roseum and related species (
33). The formation of 3PG-F
420-0, however, does not require any reduction step. Instead, enzyme assays revealed that 3-phospho-
d-glycerate (3-PG) is activated by CofC, presumably forming the short-lived intermediate 3-(guanosine-5′-diphospho)-
d-glycerate (GPPG), which is further transferred to the F
O core by the action of CofD.
However, it remained elusive, which amino acid residues within the CofC protein conferred the specificity switch toward 3-PG and how genetic mutation might have led to the evolution of 3PG-F
420 biosynthesis. Furthermore, the question persisted, why the CofC/CofD reaction only proceeds as a combined reaction and how reactive intermediates like LPPG are stabilized. Another open question concerned the role of 2-PL in the biosynthesis of F
420 in archaea. While our previous data (
22) matched seminal observations (
29) of a substantial turnover of 2-PL by CofC enzymes of archaeal origin, other studies raised doubts that 2-PL is a genuine substrate of archaeal CofC homologs (
32).
Here, we present a crystal structure of the 3-PG activating CofC from Mycetohabitans sp. B3 and revealed the amino acid residues governing 3-PG activation. By site-directed mutagenesis, we shed light on the evolution of 3PG-F420. Furthermore, we bring to attention that CofC strongly binds its product GPPG and collaborates closely with its partner CofD to control the flux of intermediates into the F420 biosynthesis pathway.