Structure of an amide bond forming F(420):gamma-glutamyl ligase from Archaeoglobus fulgidus -- a member of a new family of non-ribosomal peptide synthases.
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:gamma-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:gamma-glutamyl ligase (CofE-AF) from Archaeoglobus fulgidus and its complex with GDP at 2.5 A and 1.35 A 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.
Midwest Center for Structural Genomics and Structural Biology Center, Biosciences, Argonne National Laboratory, Argonne, IL 60439, USA.
B
B
Nocek
E
E
Evdokimova
M
M
Proudfoot
M
M
Kudritska
L L
LL
Grochowski
R H
RH
White
A
A
Savchenko
A F
AF
Yakunin
A
A
Edwards
A
A
Joachimiak
D000577:Amides / Q000378:metabolism*; D000595:Amino Acid Sequence; D019611:Archaeoglobus fulgidus / Q000201:enzymology*; D001665:Binding Sites; D003067:Coenzymes / Q000096:biosynthesis / Q000378:metabolism*; D019281:Dimerization; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000577:Amides; D003067:Coenzymes; D008670:Metals; D009711:Nucleotides; D011994:Recombinant Proteins; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase