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- PDB-7ly5: Proteolyzed crystal structure of the bacillamide NRPS, BmdB, in c... -

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Basic information

Entry
Database: PDB / ID: 7ly5
TitleProteolyzed crystal structure of the bacillamide NRPS, BmdB, in complex with the oxidase BmdC
Components
  • BmdB, Bacillamide NRPS
  • BmdC, oxidase
KeywordsBIOSYNTHETIC PROTEIN/OXIDOREDUCTASE / Nonribosomal peptide synthetases / Bacillamide / FLAVOPROTEIN / BIOSYNTHETIC PROTEIN / BIOSYNTHETIC PROTEIN-OXIDOREDUCTASE complex
Function / homologyFLAVIN MONONUCLEOTIDE
Function and homology information
Biological speciesThermoactinomyces vulgaris (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.5 Å
AuthorsFortinez, C.M. / Schmeing, T.M.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)FDN-148472 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 1.2Apr 3, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: BmdC, oxidase
A: BmdB, Bacillamide NRPS
hetero molecules


Theoretical massNumber of molelcules
Total (without water)55,7353
Polymers55,2792
Non-polymers4561
Water2,144119
1
B: BmdC, oxidase
A: BmdB, Bacillamide NRPS
hetero molecules

B: BmdC, oxidase
A: BmdB, Bacillamide NRPS
hetero molecules


Theoretical massNumber of molelcules
Total (without water)111,4706
Polymers110,5584
Non-polymers9132
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation18_444-x-2/3,-x+y-1/3,-z-1/31
Unit cell
Length a, b, c (Å)85.524, 85.524, 415.188
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number155
Space group name H-MH32
Space group name HallR32"
Symmetry operation#1: x,y,z
#2: -y,x-y,z
#3: -x+y,-x,z
#4: x-y,-y,-z
#5: -x,-x+y,-z
#6: y,x,-z
#7: x+1/3,y+2/3,z+2/3
#8: -y+1/3,x-y+2/3,z+2/3
#9: -x+y+1/3,-x+2/3,z+2/3
#10: x-y+1/3,-y+2/3,-z+2/3
#11: -x+1/3,-x+y+2/3,-z+2/3
#12: y+1/3,x+2/3,-z+2/3
#13: x+2/3,y+1/3,z+1/3
#14: -y+2/3,x-y+1/3,z+1/3
#15: -x+y+2/3,-x+1/3,z+1/3
#16: x-y+2/3,-y+1/3,-z+1/3
#17: -x+2/3,-x+y+1/3,-z+1/3
#18: y+2/3,x+1/3,-z+1/3

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Components

#1: Protein BmdC, oxidase


Mass: 23143.449 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)
#2: Protein BmdB, Bacillamide NRPS


Mass: 32135.408 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 / Flavin mononucleotide


Mass: 456.344 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H21N4O9P / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 119 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.64 Å3/Da / Density % sol: 53.46 %
Crystal growTemperature: 295.15 K / Method: vapor diffusion, sitting drop
Details: 50mM Tris-HCl pH7.5, 160mM KCl and 21% PEG3350 (wt/vol)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.979 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Feb 9, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 2.5→46.3 Å / Num. obs: 20730 / % possible obs: 99.7 % / Redundancy: 11.7 % / Biso Wilson estimate: 53.54 Å2 / Rpim(I) all: 0.033 / Rrim(I) all: 0.111 / Net I/σ(I): 29.5
Reflection shellResolution: 2.5→2.54 Å / Mean I/σ(I) obs: 2.6 / Num. unique obs: 20730 / CC1/2: 0.933

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Processing

Software
NameVersionClassification
PHENIX1.15.2_3472refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: BmdC, oxidase

Resolution: 2.5→46.3 Å / SU ML: 0.3077 / Cross valid method: FREE R-VALUE / σ(F): 1.39 / Phase error: 26.4027
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2551 1999 9.65 %
Rwork0.2286 18714 -
obs0.2312 20713 99 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 65.77 Å2
Refinement stepCycle: LAST / Resolution: 2.5→46.3 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3553 0 31 119 3703
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00683653
X-RAY DIFFRACTIONf_angle_d0.88784968
X-RAY DIFFRACTIONf_chiral_restr0.0552567
X-RAY DIFFRACTIONf_plane_restr0.0045642
X-RAY DIFFRACTIONf_dihedral_angle_d20.58591312
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.5-2.560.30481270.27611195X-RAY DIFFRACTION90.49
2.56-2.630.26661410.25891306X-RAY DIFFRACTION98.77
2.63-2.710.28381410.24511320X-RAY DIFFRACTION99.73
2.71-2.80.30691420.24071324X-RAY DIFFRACTION99.86
2.8-2.90.32441410.2491327X-RAY DIFFRACTION99.73
2.9-3.010.2881420.25791333X-RAY DIFFRACTION99.66
3.01-3.150.2691430.24321343X-RAY DIFFRACTION99.93
3.15-3.310.27441420.25491326X-RAY DIFFRACTION99.93
3.31-3.520.26131430.22781343X-RAY DIFFRACTION99.87
3.52-3.790.23381430.22221333X-RAY DIFFRACTION99.06
3.79-4.180.21351440.20491347X-RAY DIFFRACTION99.87
4.18-4.780.20191460.18551370X-RAY DIFFRACTION99.93
4.78-6.020.2291480.22711384X-RAY DIFFRACTION100
6.02-46.30.29961560.24241463X-RAY DIFFRACTION99.14

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