Volume 10, Issue 3, Pages 301-310 (March 2002) Structural Basis for the Cyclization of the Lipopeptide Antibiotic Surfactin by the Thioesterase Domain SrfTE  Steven D Bruner, Thomas Weber, Rahul M Kohli, Dirk Schwarzer, Mohamed A Marahiel, Christopher T Walsh, Milton T Stubbs  Structure  Volume 10, Issue 3, Pages 301-310 (March 2002) DOI: 10.1016/S0969-2126(02)00716-5

Figure 1 Surfactin Biosynthesis by the Modular Peptide Synthetase (A) The srf operon (top) with the three genes srfA-A, srfA-B, and srfA-C coding for the surfactin synthetase subunits shown below the genes. Bars indicate the positions of modules within the protein, whereas the individual domains are shown as colored balls: A, adenylation domain; PCP, peptidyl carrier protein domain; C, condensation domain; E, epimerization domain; TE, thioesterase domain. The 4′-phosphopantetheinyl cofactors with their active thiol groups are shown with the corresponding peptides attached at their current synthesis states. The growing peptide chain is passed from left to right, until the linear product at the last PCP domain is cyclized to the lipopeptide by the TE domain. (B) The SNAC (S-N-acetyl cysteamine) acyl peptide mimic can be cyclized by the genetically excised SrfTE domain. The native peptide (R = DLeu) and the soluble substrate (R = DOrn) are illustrated. The β-hydroxy fatty acid (FA) is likely to be attached by the N-terminal C domain. Structure 2002 10, 301-310DOI: (10.1016/S0969-2126(02)00716-5)

Figure 2 Overall Architecture of the Thioesterase Domain of Surfactin Synthetase (A) Ribbon representation of the dimer observed in the asymmetric unit (noncrystallographic diad axis vertical; helices, red; β sheets, blue). The left monomer represents the `C' (`closed') conformation of the active site and the right monomer represents the `O' (`open') conformation. The lid region (αL1, αL2, and αL3), in green, surrounds the peptide binding pocket. The catalytic triad residues (Ser80, His207, and Asp107) are rendered as ball and stick models in gold. An unordered loop region of monomer `C' is shown as a dotted line. (B and C) Schematic diagram of the general secondary structure of the α/β hydrolyase family of enzymes (B) and surfactin thioesterase domain (C). Sheets, blue arrows; helices, red cylinders. The positions of the three conserved catalytic triad residues are indicated. Structure 2002 10, 301-310DOI: (10.1016/S0969-2126(02)00716-5)

Figure 3 Alignment of Peptide Cyclizing Thioesterase Domains from B. brevis Gramicidin Synthetase and Tyrocidine Synthetase with the Crystallized SrfTE Numbering refers to the SrfTE construct; TE domain sequences of GrsTE [36, 37] and TycTE [38] as published. The positions of the catalytic triad residues are indicated with asterisks. The secondary structure coloring scheme is the same as in Figure 2A. Conserved residues, orange; partially conserved residues, yellow. Structure 2002 10, 301-310DOI: (10.1016/S0969-2126(02)00716-5)

Figure 4 Differences in the Active Site Conformation between Observed Monomers Ribbon representation of the superposition of the two monomers observed in the crystallographic dimer. Ribbon coloring is the same as in Figure 2A, except that the lid helices of the `O' (`open') conformer are shown in green and those of the `C' (`closed') conformer are shown in yellow. The positions of the catalytic triad (gold) and an observed sulfate ion (magenta) are rendered in ball and stick representation. Structure 2002 10, 301-310DOI: (10.1016/S0969-2126(02)00716-5)

Figure 5 Peptide Binding Region of the Open Conformer (A) Stereodiagram of electron density maps of the active site region of the `O' (`open') conformer. |2Fo − Fc| map (blue) contoured at 1.0 σ and an |Fo − Fc| density map (yellow) contoured at 2.5 σ illustrate the partially ordered substrate peptide density. Protein residues, white; modeled leucine amino acid, red. (B) Surface representation of the peptide binding region. Amino acid side chains lining the active site bowl, red; catalytic triad residues, yellow. Structure 2002 10, 301-310DOI: (10.1016/S0969-2126(02)00716-5)

Figure 6 Model for Thioesterase Catalyzed Peptide Cyclization (A) Docking of PCP domain (yellow) to Srf TE (red and blue). Phosphopantetheine (pan), in green, is modeled along a cleft from the observed sulfate ion to the active site. The position of the pan attachment site on a flexible loop of the PCP is indicated with an asterisk. (B) Accessible surface representation of the SrfTE showing a modeled pan arm and a five-amino acid peptide in licorice representation. The proposed PCP interaction surface is tinted yellow. Structure 2002 10, 301-310DOI: (10.1016/S0969-2126(02)00716-5)

Figure 7 The β-Hydroxy-Acyl-Heptapeptidyl-O-Ser TE Acyl Enzyme Intermediate Is Schematized in the Hydrophobic Active Site Bowl Intramolecular deacylation by the β hydroxyl releases the macrolactonized surfactin from its covalent tethering to the TE domain (R, fatty acid chain). Structure 2002 10, 301-310DOI: (10.1016/S0969-2126(02)00716-5)