Structural and Functional Insights into a Peptide Bond-Forming Bidomain from a Nonribosomal Peptide Synthetase

Abstract

The crystal structure of the bidomain PCP-C from modules 5 and 6 of the nonribosomal tyrocidine synthetase TycC was determined at 1.8 A resolution. The bidomain structure reveals a V-shaped condensation domain, the canyon-like active site groove of which is associated with the preceding peptidyl carrier protein (PCP) domain at its donor side. The relative arrangement of the PCP and the peptide bond-forming condensation (C) domain places the active sites approximately 50 A apart. Accordingly, this PCP-C structure represents a conformational state prior to peptide transfer from the donor-PCP to the acceptor-PCP domain, implying the existence of additional states of PCP-C domain interaction during catalysis. Additionally, PCP-C exerts a mode of cyclization activity that mimics peptide bond formation catalyzed by C domains. Based on mutational data and pK value analysis of active site residues, it is suggested that nonribosomal peptide bond formation depends on electrostatic interactions rather than on general acid/base catalysis.

Use of Limited Proteolysis and Mutagenesis To Identify Folding Domains and Sequence Motifs Critical for Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase Activity

“…2A. For the seven studied proteins, the same predicted structural homologs were found, including surfactin A synthetase (PDB ID 2VSQ [45]), tyrocidine synthetase TycC (PDB ID 2JGP [40]), VibH, a nonribosomal peptide synthetase (NRPS) condensation enzyme (PDB ID 1L5A [46]), polyketide synthase-associated protein 5 (PDB ID 1Q9J [47]), trichothecene 3-o-acetyltransferase (PDB ID 3B30 [48]), or vinorine synthase (PDB ID 2BGH [49]). All the found homologs were CoA-dependent acyltransferases containing the HHxxxDG motif also present in WS/DGAT.…”

“…The Ma2 3D structure was predicted by homology modeling using the Phyre server (38) (http://www.sbg.bio.ic.ac.uk /phyre2) or the robetta server (39) (http://robetta.bakerlab.org/). The crystal structure of tyrocidine synthetase TycC (PDB identifier [ID] 2JGP) was used as the template (40). An image of the resulting 3D model was generated using the program Pymol (DeLano Scientific, Palo Alto, CA, USA) (http://www.pymol.org).…”

New Structural Data Reveal the Motion of Carrier Proteins in Nonribosomal Peptide Synthesis

“…Strikingly, a simple rotation of the PCP domain appears to be sufficient to deliver the substrate from the C domain to the TE domain, and this could be accomplished without major structural rearrangement of the NRPS. Furthermore, a PCP/C di‐domain structure revealed the upstream PCP domain located close to the donor binding site of the C domain, but with the Ppant attachment site rotated away by 180° when the PCP domain is not loaded 47. This closely resembles the interaction between the PCP and TE domains in the AB3403 structure 26.…”

Biosynthesis of Novel Pyoverdines by Domain Substitution in a Nonribosomal Peptide Synthetase of Pseudomonas aeruginosa

“…One possible explanation for this is that C domains in a new context may be unable to receive some incoming peptides due to steric constraints. The crystal structures of C domains show the catalytic center covered by a lid region within a channel between two subdomains (11,(29)(30)(31), and these features may contribute to some incoming peptide chains being physically blocked from reaching the catalytic center. It is also important to note that the previous in vitro studies of C domain donor site specificity focused on single residues (9, 10), or at most a tetrapeptide (28), rather than the longer peptide chains that substituted C domains further down an NRPS assembly line might encounter.…”