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- PDB-7ly6: Structure of a trans-acting NRPS oxidase, BmdC, involved in bacil... -

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Entry
Database: PDB / ID: 7ly6
TitleStructure of a trans-acting NRPS oxidase, BmdC, involved in bacillamide biosynthesis
ComponentsBmdC, NRPS oxidase
KeywordsFLAVOPROTEIN / Nonribosomal peptide synthetases Bacillamide
Function / homologyFLAVIN MONONUCLEOTIDE / GLYCINE / PHOSPHATE ION
Function and homology information
Biological speciesThermoactinomyces vulgaris (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.73 Å
AuthorsFortinez, C.M. / Bloudoff, K. / 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 1.2Oct 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

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Assembly

Deposited unit
A: BmdC, NRPS oxidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)38,8044
Polymers38,1771
Non-polymers6263
Water1,820101
1
A: BmdC, NRPS oxidase
hetero molecules

A: BmdC, NRPS oxidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)77,6078
Polymers76,3552
Non-polymers1,2536
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation14_555-x,-y+1/2,z1
Buried area14480 Å2
ΔGint-107 kcal/mol
Surface area26860 Å2
MethodPISA
Unit cell
Length a, b, c (Å)188.150, 188.150, 188.150
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number199
Space group name H-MI213
Space group name HallI2b2c3
Symmetry operation#1: x,y,z
#2: z,x,y
#3: y,z,x
#4: -y,-z+1/2,x
#5: z,-x,-y+1/2
#6: -y+1/2,z,-x
#7: -z,-x+1/2,y
#8: -z+1/2,x,-y
#9: y,-z,-x+1/2
#10: x,-y,-z+1/2
#11: -x+1/2,y,-z
#12: -x,-y+1/2,z
#13: x+1/2,y+1/2,z+1/2
#14: z+1/2,x+1/2,y+1/2
#15: y+1/2,z+1/2,x+1/2
#16: -y+1/2,-z+1,x+1/2
#17: z+1/2,-x+1/2,-y+1
#18: -y+1,z+1/2,-x+1/2
#19: -z+1/2,-x+1,y+1/2
#20: -z+1,x+1/2,-y+1/2
#21: y+1/2,-z+1/2,-x+1
#22: x+1/2,-y+1/2,-z+1
#23: -x+1,y+1/2,-z+1/2
#24: -x+1/2,-y+1,z+1/2

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Components

#1: Protein BmdC, NRPS oxidase


Mass: 38177.254 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: Chemical ChemComp-GLY / GLYCINE / Glycine


Type: peptide linking / Mass: 75.067 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H5NO2
#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: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 101 / 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

Crystal growTemperature: 295.15 K / Method: vapor diffusion, sitting drop
Details: 35% PEG1500 (wt/vol), 0.1M SPG buffer pH9.0. [1M SPG buffer: (1.48g of succinic acid, 6.04g sodium dihydrogen phosphate monohydrate and 3.28g of glycine in 100ml of water)]

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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: Oct 6, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 2.73→94.08 Å / Num. obs: 29493 / % possible obs: 99.9 % / Redundancy: 9.2 % / Biso Wilson estimate: 55.83 Å2 / Rmerge(I) obs: 0.249 / Net I/σ(I): 1.6
Reflection shellResolution: 2.73→2.86 Å / Num. unique obs: 29506 / CC1/2: 0.354

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Processing

Software
NameVersionClassification
PHENIX1.15.2_3472refinement
iMOSFLMdata reduction
Aimlessdata scaling
MoRDaphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3EO7

3eo7


Resolution: 2.73→94.07 Å / SU ML: 0.3065 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 18.928
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.1944 1553 5.27 %
Rwork0.1719 27939 -
obs0.1731 29492 99.98 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 70.33 Å2
Refinement stepCycle: LAST / Resolution: 2.73→94.07 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2599 0 41 101 2741
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01222701
X-RAY DIFFRACTIONf_angle_d1.24683662
X-RAY DIFFRACTIONf_chiral_restr0.0574394
X-RAY DIFFRACTIONf_plane_restr0.0075468
X-RAY DIFFRACTIONf_dihedral_angle_d19.8886983
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.73-2.820.29611440.31012528X-RAY DIFFRACTION100
2.82-2.920.30911380.27212512X-RAY DIFFRACTION100
2.92-3.040.29621480.24612498X-RAY DIFFRACTION100
3.04-3.180.24871560.23222493X-RAY DIFFRACTION100
3.18-3.340.24681450.20112543X-RAY DIFFRACTION100
3.34-3.550.22661490.18452518X-RAY DIFFRACTION100
3.55-3.830.17771290.15272532X-RAY DIFFRACTION100
3.83-4.210.14061550.13222521X-RAY DIFFRACTION100
4.21-4.820.14611450.10912555X-RAY DIFFRACTION100
4.82-6.070.1583990.14352604X-RAY DIFFRACTION100
6.07-94.070.17661450.18352635X-RAY DIFFRACTION99.78
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)
10.0858970256010.008365907724810.03227701672740.0141027429461-0.02477551857090.0296709091782-0.331559278915-0.0673044205654-0.09818464692890.322216859680.356693590992-0.193138884011-0.097332713885-0.07530071795531.84064724282E-90.4159082388390.170647811162-0.1227459113470.2559089330350.01161173686390.42998389028511.23643.11848.898
20.00358614928936-0.0341090180693-0.02328416899330.03515908684550.02487142157780.08874525216120.3169262378470.00934438203894-0.02116879593480.122484787614-0.0670474902790.07711216643360.2282359122110.01226110652751.77262604614E-80.2323904037710.123349708603-0.1334632958250.2934359206770.008624492742770.448510580184-9.30335.59624.625
3-0.00876048652996-0.03888093644540.0114540889989-0.04790281843110.01277615154060.00662973956466-0.2822726046290.244137532807-0.0961475105498-0.5624994445150.2250275031760.1118409276840.1432090761050.00795075370734-1.04766160433E-70.4747557782690.2277780068910.06331910500470.677863350863-0.1759615741540.5055941243311.8136.704-0.001
40.431413951560.09257552084880.08650073679310.112003841762-0.02249134559470.1368960945610.1639483855630.141921956721-0.07942618355280.0405283081739-0.0884774246726-0.0951971946737-0.1002002059380.139613165642-4.05688659835E-100.1689645502470.2358360958270.06101397438030.421292040513-0.03042314873420.36564400791313.14544.12312.978
50.00318807064156-0.001341501153180.002285535190680.0001053721069160.002643738212370.00595219079101-0.01260452866950.0685674749205-0.0407065241613-0.0107246542895-0.02825725805050.01059251461870.0172897444139-0.008041872925875.80550302771E-60.4969292145540.0733186248997-0.04935898827640.648099732805-0.04957187062320.7155961403390.24934.00312.443
61.999985673462.000015579081.99997573892.000009012011.999995635521.99999484024-0.61719729688-0.228879325821-0.6728267686540.04793494022650.31864849148-0.9174292697330.5348477552441.14957349140.2976834974391.66103209857-0.0635797041880.02825531075351.520615373160.1215349681741.5886161962-2.12228.52813.855
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1( CHAIN A AND RESID 4:75 )A4 - 75
2X-RAY DIFFRACTION2( CHAIN A AND RESID 76:130 )A76 - 130
3X-RAY DIFFRACTION3( CHAIN A AND RESID 131:160 )A131 - 160
4X-RAY DIFFRACTION4( CHAIN A AND RESID 161:324 )A161 - 324
5X-RAY DIFFRACTION5( CHAIN A AND RESID 402:402 )A402
6X-RAY DIFFRACTION6( CHAIN A AND RESID 403:403 )A403

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