Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S

• Published in: The EMBO journal 1997-07, Vol.16 (14), p.4174-4183
• Main Authors: Conti, Elena, Stachelhaus, Torsten, Marahiel, Mohamed A., Brick, Peter
• Format: Article
• Language: English
• Subjects: Adenosine Monophosphate - chemistry; Adenosine Monophosphate - metabolism; Amino Acid Isomerases - chemistry; Amino Acid Isomerases - metabolism; Amino Acid Sequence; Animals; Bacillus - chemistry; Bacillus - enzymology; Bacillus brevis; Binding Sites; Coleoptera - enzymology; Crystallography, X-Ray; Gramicidin - biosynthesis; Hydrogen Bonding; Luciferases - chemistry; Models, Molecular; Molecular Sequence Data; non-ribosomal peptide biosynthesis; peptide synthetases; Phenylalanine - chemistry; Phenylalanine - metabolism; Protein Binding; Protein Conformation; Protein Structure, Secondary; Sequence Alignment; X-ray crystallography
• ISSN: 0261-4189; 1460-2075; 1460-2075
• DOI: 10.1093/emboj/16.14.4174

The non‐ribosomal synthesis of the cyclic peptide antibiotic gramicidin S is accomplished by two large multifunctional enzymes, the peptide synthetases 1 and 2. The enzyme complex contains five conserved subunits of ∼60 kDa which carry out ATP‐dependent activation of specific amino acids and share extensive regions of sequence similarity with adenylating enzymes such as firefly luciferases and acyl‐CoA ligases. We have determined the crystal structure of the N‐terminal adenylation subunit in a complex with AMP and L‐phenylalanine to 1.9 Å resolution. The 556 amino acid residue fragment is folded into two domains with the active site situated at their interface. Each domain of the enzyme has a similar topology to the corresponding domain of unliganded firefly luciferase, but a remarkable relative domain rotation of 94° occurs. This conformation places the absolutely conserved Lys517 in a position to form electrostatic interactions with both ligands. The AMP is bound with the phosphate moiety interacting with Lys517 and the hydroxyl groups of the ribose forming hydrogen bonds with Asp413. The phenylalanine substrate binds in a hydrophobic pocket with the carboxylate group interacting with Lys517 and the α‐amino group with Asp235. The structure reveals the role of the invariant residues within the superfamily of adenylate‐forming enzymes and indicates a conserved mechanism of nucleotide binding and substrate activation.