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- PDB-7ly7: Crystal structure of the elongation module of the bacillamide NRP... -

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Basic information

Entry
Database: PDB / ID: 7ly7
TitleCrystal 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 Bacillamide / FLAVOPROTEIN
Function / homologyFLAVIN MONONUCLEOTIDE / Chem-YOA
Function and homology information
Biological speciesThermoactinomyces vulgaris (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.8 Å
AuthorsFortinez, C.M. / Sharon, I. / 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 2.0Aug 3, 2022Group: Advisory / Derived calculations ...Advisory / Derived calculations / Non-polymer description / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_validate_close_contact / struct_conn
Item: _chem_comp.formula / _chem_comp.formula_weight / _entity.formula_weight
Revision 2.1Oct 18, 2023Group: 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

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)156,5414
Polymers155,2952
Non-polymers1,2462
Water0
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)313,0828
Polymers310,5894
Non-polymers2,4924
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation8_555x-y,-y,-z1
Unit cell
Length a, b, c (Å)149.960, 149.960, 319.551
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number179
Space group name H-MP6522
Space group name HallP652(x,y,z+1/12)
Symmetry operation#1: x,y,z
#2: x-y,x,z+5/6
#3: y,-x+y,z+1/6
#4: -y,x-y,z+2/3
#5: -x+y,-x,z+1/3
#6: x-y,-y,-z
#7: -x,-x+y,-z+1/3
#8: -x,-y,z+1/2
#9: y,x,-z+2/3
#10: -y,-x,-z+1/6
#11: -x+y,y,-z+1/2
#12: x,x-y,-z+5/6

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Components

#1: Protein BmdC, Oxidase


Mass: 38049.125 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: 117245.453 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
#4: Chemical ChemComp-YOA / 5'-{[(2R,3S)-3-amino-2-({2-[(N-{(2R)-4-[(dihydroxyphosphanyl)oxy]-2-hydroxy-3,3-dimethylbutanoyl}-beta-alanyl)amino]ethyl}sulfanyl)-4-sulfanylbutane-1-sulfonyl]amino}-5'-deoxyadenosine


Mass: 789.839 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C25H44N9O12PS3 / Feature type: SUBJECT OF INVESTIGATION
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: 3.34 Å3/Da / Density % sol: 63.17 %
Crystal growTemperature: 295.15 K / Method: vapor diffusion, sitting drop
Details: 0.2M sodium citrate, 14% (w/v) PEG3350, 0.08M guanidine hydrochloride, 0.1M Bis Tris propane pH6.1 and 5.6mg/ml of the dimeric complex of the elongation module of BmdB and the oxidase, BmdC

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: CLSI / Beamline: 08ID-1 / Wavelength: 0.979 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Sep 7, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 3.8→82.36 Å / Num. obs: 21691 / % possible obs: 100 % / Redundancy: 23.3 % / Biso Wilson estimate: 90.34 Å2 / CC1/2: 0.996 / Net I/σ(I): 4.5
Reflection shellResolution: 3.8→4.1 Å / Mean I/σ(I) obs: 1.5 / Num. unique obs: 21691 / CC1/2: 0.473

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Processing

Software
NameVersionClassification
PHENIX1.15.2_3472refinement
MOSFLMdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5T3E, 7LY6, 7LY5

5t3e

7ly6

7ly5


Resolution: 3.8→49.28 Å / SU ML: 0.7435 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 34.2492
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.3259 2172 10.01 %
Rwork0.2979 19519 -
obs0.3006 21691 99.86 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 182.85 Å2
Refinement stepCycle: LAST / Resolution: 3.8→49.28 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10002 0 0 0 10002
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.003410241
X-RAY DIFFRACTIONf_angle_d0.739713914
X-RAY DIFFRACTIONf_chiral_restr0.04651526
X-RAY DIFFRACTIONf_plane_restr0.00411800
X-RAY DIFFRACTIONf_dihedral_angle_d13.18013803
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.8-3.880.44711330.4181197X-RAY DIFFRACTION99.92
3.88-3.970.5341320.5291165X-RAY DIFFRACTION98.41
3.97-4.070.41031320.37411187X-RAY DIFFRACTION100
4.07-4.180.39751320.35451199X-RAY DIFFRACTION99.7
4.18-4.310.40111320.32911188X-RAY DIFFRACTION100
4.31-4.440.31811340.32351181X-RAY DIFFRACTION99.85
4.44-4.60.34731320.2861199X-RAY DIFFRACTION100
4.6-4.790.32931350.27051213X-RAY DIFFRACTION100
4.79-50.30271350.27261218X-RAY DIFFRACTION99.93
5-5.270.31781320.27481196X-RAY DIFFRACTION100
5.27-5.60.30061350.26921225X-RAY DIFFRACTION99.93
5.6-6.030.31571370.29411218X-RAY DIFFRACTION100
6.03-6.630.29151360.27371236X-RAY DIFFRACTION100
6.63-7.590.29861400.26181246X-RAY DIFFRACTION100
7.59-9.550.26761410.25341276X-RAY DIFFRACTION100
9.55-49.280.2711540.25131375X-RAY DIFFRACTION100
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.39207779438-0.903201743331-0.1971596840171.270289153970.1274898919150.1947952319040.001854617869682.18358469747-0.163796607722-0.2623990636061.22942517732.14249163859-0.40100286448-1.01047523344-0.4263003573681.757362429421.26255864968-0.2414426509213.03773209694-1.224747563293.03316031416-22.511719266411.5115263878-5.76041451954
24.670485102890.1906231308280.3748300023176.44404465566-0.186978583723.886243199880.256903888110.07854698681140.07291157420120.116748227088-0.8950654702780.408568797684-0.76465037603-0.2193296463560.5868982579430.7570440737410.0933860052989-0.07802059312740.673897804276-0.06957824284460.39281443405512.89618177439.46573452492-0.0314957864249
30.05868418578520.9407250367230.3441247918160.800832561582-0.3676719330431.092257864110.3175624504960.4177706544252.067174453640.760289229449-0.2509727638130.7638813077160.003450857140660.698598225574-0.01609280792825.95316780742-0.304632294911-0.3448342168372.03378188937-0.5092253053163.5250505843711.2830612037111.67139054617.505332575
42.609458537010.9980268761210.6360326059411.6671943039-0.1325643610760.630928934965-1.014117384610.454323744480.09827974362351.15736327458-0.7781867851611.99926655009-0.7535179986383.12630827729-0.009452230249286.20499190863-0.210792201191-0.8884507034822.84914962357-0.3703562694533.6490909858214.0222515505116.14030883414.2425112526
50.270585533744-0.181304021297-0.2377862432210.8035417299730.1126102741560.0360659127612-0.2282398082960.2932858332320.6692380196792.285693596420.3458056483611.16010305238-0.7165007405330.02840852555170.05925434023133.168769172920.233050045707-0.2134556460512.41768367115-0.2324053301292.51032113005-9.81758739964108.5906746097.31273145324
62.51939036081-1.831689058950.1517926192622.73017752230.3941123296011.17968553930.198053515195-0.5004953219341.143274715850.3831559128350.221143853631-0.63077611225-1.166418625780.135259331471-0.7801533472912.455043303130.0967714979343-0.3621891131731.11778286321-0.1666597074682.297179979541.91304536707100.293055276-3.70011944503
73.675023185610.164185798402-0.5071386913833.737156807271.521081355475.270166173490.04446739849650.0234652792420.5203397839570.554121433001-0.356527494027-0.2490238536-0.0180394234771-0.4580479291870.2378238937530.8770894702740.54989471847-0.5321136720390.628638205769-0.235762103431.10235256343-0.10024517966844.3072800724-18.6582710488
81.794847980780.11774558047-0.6418010157754.13878989787-0.4864773650025.78245416351-0.355266864599-2.675998518130.5339336118781.40785244495-0.9228067641720.223280745173-1.22443328595-1.709291231490.7503058552711.811205775531.22230408883-0.7684047477371.3185057503-0.4223630304821.93690950323-6.0052436408958.0460057674-3.78096861966
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'B' and (resid 2 through 73 )
2X-RAY DIFFRACTION2chain 'B' and (resid 74 through 330 )
3X-RAY DIFFRACTION3chain 'A' and (resid 847 through 950 )
4X-RAY DIFFRACTION4chain 'A' and (resid 951 through 1001 )
5X-RAY DIFFRACTION5chain 'A' and (resid 1002 through 1055 )
6X-RAY DIFFRACTION6chain 'A' and (resid 1056 through 1288 )
7X-RAY DIFFRACTION7chain 'A' and (resid 1289 through 1580 )
8X-RAY DIFFRACTION8chain 'A' and (resid 1581 through 1901)

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