Cryo-EM Structure of a Monomeric Safracin B Nonribosomal Peptide Synthetase Enabled by an MbtH-like Protein and Covalent Probe Trapping

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Abstract
Nonribosomal peptide synthetases (NRPSs) assemble structurally complex natural products but remain challenging targets for high-resolution structural analysis because of their conformational heterogeneity and dynamic catalytic cycles. SacC/SfmC is an unusual single-module NRPS that diverges from canonical peptide-forming assembly lines and instead catalyzes two sequential Pictet–Spengler reactions between a tyrosine-derived unit and a peptidyl aldehyde generated through thioester reduction. This cascade forms two C–C bonds, stereoselectively installs multiple stereogenic centers, and constructs the pentacyclic scaffold of bis-tetrahydroisoquinoline antitumor alkaloids including safracin B and the clinically important ecteinascidins. Here, we establish a state-resolved cryo-electron microscopy (cryo-EM) strategy for structural interrogation of the monomeric noncanonical NRPS SacC and report its thioesterification-state structure at 2.76 Å resolution. Stabilization of this transient catalytic state was achieved by combining the cognate MbtH-like partner protein with a tailored covalent adenosine vinylsulfonamide probe that mimics the aminoacyl-adenylate intermediate and traps the phosphopantetheine-tethered species within the adenylation domain. The structure directly visualizes a carrier protein-tethered intermediate and reveals the spatial organization of the Pictet–Spengler, adenylation, peptidyl carrier protein, and reduction domains, providing the first structural view of a Pictet–Spengler cyclization domain embedded within an NRPS assembly line. In parallel, nonreducible ester probes corresponding to downstream PCP-bound intermediates were developed to enable structural interrogation of additional catalytic states along the SacC reaction trajectory. These results demonstrate that rational chemical probe design enables state-resolved cryo-EM analysis of highly dynamic, single-module NRPSs and establishes a general strategy for capturing catalytic trajectories and enabling the engineering of noncanonical NRPS reactivity.
Funding Information
  • Naito Foundation
  • Institute for Fermentation, Osaka
  • Deutscher Akademischer Austauschdienst (57610340)
  • Toyota Physical and Chemical Research Institute
  • Japan Agency for Medical Research and Development (JP223fa627001)
  • Japan Agency for Medical Research and Development (JP25ama121038)
  • Asahi Glass Foundation
  • Japan Foundation for Applied Enzymology
  • Japan Society for the Promotion of Science (JP17KK0141)
  • Japan Society for the Promotion of Science (JP21H05260)
  • Japan Society for the Promotion of Science (JP22H00346)
  • Japan Society for the Promotion of Science (JP22H05127)
  • Japan Society for the Promotion of Science (JP22K14790)
  • Japan Society for the Promotion of Science (JP23H05470)
  • Japan Society for the Promotion of Science (JP23KJ0690)
  • Japan Society for the Promotion of Science (JP24K08726)
  • Japan Society for the Promotion of Science (JPJSBP120223501)
  • Japan Science and Technology Agency (JPMJAX211A)

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