Elongation of the Poly-γ-glutamate Tail of F420 Requires Both Domains of the F420:γ-Glutamyl Ligase (FbiB) of Mycobacterium tuberculosis

Abstract

Cofactor F 420 is an electron carrier with a major role in the oxidoreductive reactions of Mycobacterium tuberculosis, the causative agent of tuberculosis. A ␥-glutamyl ligase catalyzes the final steps of the F 420 biosynthesis pathway by successive additions of L-glutamate residues to F 420 -0, producing a poly-␥-glutamate tail. The enzyme responsible for this reaction in archaea (CofE) comprises a single domain and produces F 420 -2 as the major species. The homologous M. tuberculosis enzyme, FbiB, is a two-domain protein and produces F 420 with predominantly 5-7 L-glutamate residues in the poly-␥-glutamate tail. The N-terminal domain of FbiB is homologous to CofE with an annotated ␥-glutamyl ligase activity, whereas the C-terminal domain has sequence similarity to an FMN-dependent family of nitroreductase enzymes. Here we demonstrate that full-length FbiB adds multiple L-glutamate residues to F 420 -0 in vitro to produce F 420 -5 after 24 h; communication between the two domains is critical for full ␥-glutamyl ligase activity. We also present crystal structures of the C-terminal domain of FbiB in apo-, F 420 -0-, and FMN-bound states, displaying distinct sites for F 420 -0 and FMN ligands that partially overlap. Finally, we discuss the features of a full-length structural model produced by small angle x-ray scattering and its implications for the role of N-and C-terminal domains in catalysis.The cofactor F 420 is a flavin derivative that is sporadically distributed among microorganisms, mainly archaea and actinobacteria (including mycobacteria). F 420 has been emerging as a new player in the biology of mycobacteria (1), with increasing numbers of F 420 -utilizing proteins characterized from different mycobacterial species (2-8). This cofactor has been suggested to protect Mycobacterium tuberculosis, the causative agent of tuberculosis, against oxidative and nitrosative stress during pathogenesis (9 -11). At the biochemical level, cofactor F 420 functions as a hydride transfer agent in oxidoreductive reactions with a lower redox potential than that of NAD(P) ϩ (12). The biosynthesis pathway of cofactor F 420 has been investigated in both archaeal and mycobacterial species. In the current view of the proposed pathway, the first intermediate with the complete chromophore (7, ) is produced by FO synthase (FbiC in mycobacteria (13) and CofGH in archaea (14)). A transferase enzyme (FbiA in mycobacteria (15) and CofD in archaea (16)) subsequently catalyzes the addition of a 2-phospho-L-lactate moiety to FO to produce F 420 -0 (F 420 with no poly-␥-glutamate tail). The final step of the pathway is performed by a ␥-glutamyl ligase (FbiB in mycobacteria (15) and CofE in archaea (17)) that catalyzes successive additions of L-glutamate residues to F 420 -0 (Fig. 1A).The length of the poly-␥-glutamate tail varies between archaeal and mycobacterial species; in archaea, two L-glutamate residues are seen (18), whereas in mycobacteria, up to nine residues are present (3, 19). There exists an intriguing difference between the ...

The methanogenic redox cofactor F420 is widely synthesized by aerobic soil bacteria

“…Subsequently, LPPG (L-lactyl-2-diphospho-5 0 -guanosine) is proposed to be synthesized from 2-phospho-L-lactate by CofC (Grochowski et al, 2008) and transferred to F o by CofD (also known as FbiA) (Choi et al, 2001;Graupner and White, 2001;Graupner et al, 2002). The resulting LPPG sidechain is finally elongated with glutamate residues by the F 420 :γ-L-glutamyl ligase CofE (also known as FbiB) (Choi et al, 2001;Li et al, 2003;Nocek et al, 2007) that is fused with an FMNdependent oxidoreductase in Actinobacteria (Bashiri et al, 2016). For reasons still not understood, the number of glutamate residues added varies between organisms, ranging from two to three in most methanogens (Gorris and van der Drift, 1994), four to five in Methanosarcina (Gorris and van der Drift, 1994) and five to seven in Mycobacterium .…”

A revised biosynthetic pathway for the cofactor F420in bacteria

“…However, no masses corresponding to F420-0 were identified in any of the LC-MS traces from the FbiD:FbiA coupled assays, suggesting that an enzyme other than FbiD or FbiA catalyzes dehydro-F420-0 reduction. We have previously shown that full-length FbiB consists of two domains: an N-terminal domain that is homologous to the archaeal -glutamyl ligase CofE 21,33 , and a C-terminal domain of the NTR fold 34 that binds to FMN and has no known function 20 , but is essential for extending the poly--glutamate tail.…”

“…The two branches then merge at the reaction catalyzed by the transferase FbiA/CofD, where the 2-phospho-L-lactyl moiety of LPPG is transferred to Fo, forming F420-0 18,19 . Finally, the -glutamyl ligase (FbiB/CofE) catalyzes the poly-glutamylation of F420 to generate mature F420, with poly--glutamate tail lengths of ~2-8, depending on species 20,21 .…”

“…The N-terminal domain is homologous with the archaeal -glutamyl ligase CofE 33 , but is only capable of catalyzing glutamylation of F420-120 . The C-terminal domain, which binds both F420 and FMN, is essential for extending the poly--glutamate tail of F420-0 20 , but we could not explain the possible function of the FMN cofactor.…”

“…Second, the existence of FbiD has only been inferred through bioinformatics and genetic knockout studies and the enzyme has not been formally characterized 23,24 . Finally, the bacterial -glutamyl ligase FbiB is a two-domain protein 20 , in which the N-terminal domain is homologous to other F420--glutamyl ligases (including the archaeal equivalent, CofE) and the C-terminal domain adopts an FMNbinding nitroreductase (NTR) fold 20 . Although both domains are required for full -glutamyl ligase activity, no function has been associated with either the C-terminal domain or the FMN cofactor, given no redox reactions are known to be involved in F420 biosynthesis.…”