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- PDB-7ly4: Cryo-EM structure of the elongation module of the bacillamide NRP... -

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Basic information

Entry
Database: PDB / ID: 7ly4
TitleCryo-EM structure of the elongation module of the bacillamide NRPS, BmdB, in complex with the oxidase, BmdC
Components
  • BmdB, bacillamide NRPS
  • BmdC, Oxidase
KeywordsBIOSYNTHETIC PROTEIN / Nonribosomal peptide synthetases Oxidase Bacillamide synthetases
Function / homologyFLAVIN MONONUCLEOTIDE
Function and homology information
Biological speciesThermoactinomyces vulgaris (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsSharon, I. / Fortinez, C.M. / Schmeing, T.M.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: Nat Commun / Year: 2022
Title: Structures and function of a tailoring oxidase in complex with a nonribosomal peptide synthetase module.
Authors: Camille Marie Fortinez / Kristjan Bloudoff / Connor Harrigan / Itai Sharon / Mike Strauss / T Martin Schmeing /
Abstract: Nonribosomal peptide synthetases (NRPSs) are large modular enzymes that synthesize secondary metabolites and natural product therapeutics. Most NRPS biosynthetic pathways include an NRPS and ...Nonribosomal peptide synthetases (NRPSs) are large modular enzymes that synthesize secondary metabolites and natural product therapeutics. Most NRPS biosynthetic pathways include an NRPS and additional proteins that introduce chemical modifications before, during or after assembly-line synthesis. The bacillamide biosynthetic pathway is a common, three-protein system, with a decarboxylase that prepares an NRPS substrate, an NRPS, and an oxidase. Here, the pathway is reconstituted in vitro. The oxidase is shown to perform dehydrogenation of the thiazoline in the peptide intermediate while it is covalently attached to the NRPS, as the penultimate step in bacillamide D synthesis. Structural analysis of the oxidase reveals a dimeric, two-lobed architecture with a remnant RiPP recognition element and a dramatic wrapping loop. The oxidase forms a stable complex with the NRPS and dimerizes it. We visualized co-complexes of the oxidase bound to the elongation module of the NRPS using X-ray crystallography and cryo-EM. The three active sites (for adenylation, condensation/cyclization, and oxidation) form an elegant arc to facilitate substrate delivery. The structures enabled a proof-of-principle bioengineering experiment in which the BmdC oxidase domain is embedded into the NRPS.
History
DepositionMar 5, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 2, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 9, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Assembly

Deposited unit
A: BmdC, Oxidase
D: BmdC, Oxidase
E: BmdB, bacillamide NRPS
hetero molecules


Theoretical massNumber of molelcules
Total (without water)194,6415
Polymers193,7283
Non-polymers9132
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein BmdC, Oxidase


Mass: 38177.254 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoactinomyces vulgaris (bacteria) / Production host: Escherichia coli (E. coli)
#2: Protein BmdB, bacillamide NRPS


Mass: 117373.578 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoactinomyces vulgaris (bacteria) / Production host: Escherichia coli (E. coli)
#3: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H21N4O9P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Elongation module of the bacillamide NRPS, BmdB, in complex with the oxidase, BmdC
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Thermoactinomyces vulgaris (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 109 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122038 / Symmetry type: POINT

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