Structure of an Amide Bond Forming F 420:γγ-glutamyl Ligase from Archaeoglobus Fulgidus - A Member of a New Family of Non-ribosomal Peptide Synthases

Abstract

F 420 is a flavin-like redox-active coenzyme commonly used by archaea and some eubacteria in a variety of biochemical reactions in methanogenesis, the formation of secondary metabolites, the degradation of nitroaromatic compounds, activation of nitroimidazofurans, and F 420-dependent photolysis in DNA repair. Coenzyme F 420-2 biosynthesis from 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo) and lactaldehyde involves six enzymatic steps and five proteins (CofA, CofB, CofC, CofD, and CofE). CofE, a F 420-0:γ-glutamyl ligase, is responsible for the last two enzymatic steps; it catalyses the GTP-dependent addition of two L-glutamate residues to F 420-0 to form F 420-2. CofE is found in archaea, the aerobic actinomycetes, and cyanobacteria. Here, we report the first crystal structure of the apo-F 420-0:γ-glutamyl ligase (CofE-AF) from Archaeoglobus fulgidus and its complex with GDP at 2.5 Å and 1.35 Å resolution, respectively. The structure of CofE-AF reveals a novel protein fold with an intertwined, butterfly-like dimer formed by two-domain monomers. GDP and Mn 2+ are bound within the putative active site in a large groove at the dimer interface. We show that the enzyme adds a glutamate residue to both F 420-0 and F 420-1 in two distinct steps. CofE represents the first member of a new structural family of non-ribosomal peptide synthases.