title: Elongation of the Poly-γ-glutamate Tail of F420 Requires Both Domains of the F420:γ-Glutamyl Ligase (FbiB) of Mycobacterium tuberculosis.
abstract: Cofactor F420is an electron carrier with a major role in the oxidoreductive reactions ofMycobacterium tuberculosis, the causative agent of tuberculosis. A γ-glutamyl ligase catalyzes the final steps of the F420biosynthesis pathway by successive additions ofl-glutamate residues to F420-0, producing a poly-γ-glutamate tail. The enzyme responsible for this reaction in archaea (CofE) comprises a single domain and produces F420-2 as the major species. The homologousM. tuberculosisenzyme, FbiB, is a two-domain protein and produces F420with predominantly 5-7l-glutamate residues in the poly-γ-glutamate tail. The N-terminal domain of FbiB is homologous to CofE with an annotated γ-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 multiplel-glutamate residues to F420-0in vitroto produce F420-5 after 24 h; communication between the two domains is critical for full γ-glutamyl ligase activity. We also present crystal structures of the C-terminal domain of FbiB in apo-, F420-0-, and FMN-bound states, displaying distinct sites for F420-0 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.
authors: From the Structural Biology Laboratory, School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, and.
authors: Ghader
authors: G
authors: Bashiri
authors: From the Structural Biology Laboratory, School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, and.
authors: Aisyah M
authors: AM
authors: Rehan
authors: the Faculty of Medical and Health Sciences, University of Auckland, Auckland 1010, New Zealand.
authors: Sreevalsan
authors: S
authors: Sreebhavan
authors: From the Structural Biology Laboratory, School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, and.
authors: Heather M
authors: HM
authors: Baker
authors: From the Structural Biology Laboratory, School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, and.
authors: Edward N
authors: EN
authors: Baker
authors: From the Structural Biology Laboratory, School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, and c.squire@auckland.ac.nz.
authors: Christopher J
authors: CJ
authors: Squire
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D003067:Coenzymes; C511775:poly(gamma-glutamic acid); D011099:Polyglutamic Acid; D008025:Ligases
keywords: FbiB; Mycobacterium tuberculosis; cofactor F420; crystal structure; gamma-glutamyl ligase; high-performance liquid chromatography (HPLC); mass spectrometry (MS); poly-gamma-glutamate tail; x-ray crystallography