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

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.