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- PDB-6ltc: Crystal Structure of Nonribosomal peptide synthetases (NRPS), Fmo... -

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Basic information

Entry
Database: PDB / ID: 6ltc
TitleCrystal Structure of Nonribosomal peptide synthetases (NRPS), FmoA3 (S1046A)-alpha-methyl-L-serine-AMP bound form
ComponentsNonribosomal peptide synthetaseNonribosomal peptide
KeywordsBIOSYNTHETIC PROTEIN / Nonribosomal peptide synthetases (NRPS) / JBIR-34 and -35
Function / homology
Function and homology information


organic cyclic compound biosynthetic process / lipid biosynthetic process / phosphopantetheine binding / antibiotic biosynthetic process / catalytic activity
Similarity search - Function
AMP-binding / Condensation domain / Condensation domain / Amino acid adenylation domain / AMP-binding enzyme, C-terminal domain / AMP-binding enzyme C-terminal domain / AMP-binding, conserved site / Chloramphenicol acetyltransferase-like domain superfamily / Putative AMP-binding domain signature. / Polyketide synthase, phosphopantetheine-binding domain ...AMP-binding / Condensation domain / Condensation domain / Amino acid adenylation domain / AMP-binding enzyme, C-terminal domain / AMP-binding enzyme C-terminal domain / AMP-binding, conserved site / Chloramphenicol acetyltransferase-like domain superfamily / Putative AMP-binding domain signature. / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / AMP-dependent synthetase/ligase / AMP-binding enzyme, C-terminal domain superfamily / AMP-binding enzyme / Phosphopantetheine attachment site / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain
Similarity search - Domain/homology
ADENOSINE MONOPHOSPHATE / alpha-methyl-L-serine / Phenyloxazoline synthase MbtB
Similarity search - Component
Biological speciesStreptomyces sp. Sp080513GE-23 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.3 Å
AuthorsSenda, T. / Harada, A.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP17H05432 Japan
Japan Society for the Promotion of Science (JSPS)17J09750 Japan
Japan Agency for Medical Research and Development (AMED)BINDS1146 Japan
CitationJournal: Angew Chem Int Ed Engl / Year: 2021
Title: Structural and Functional Analyses of the Tridomain-Nonribosomal Peptide Synthetase FmoA3 for 4-Methyloxazoline Ring Formation.
Authors: Yohei Katsuyama / Kaoru Sone / Ayaka Harada / Seiji Kawai / Naoki Urano / Naruhiko Adachi / Toshio Moriya / Masato Kawasaki / Kazuo Shin-Ya / Toshiya Senda / Yasuo Ohnishi /
Abstract: Nonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides. FmoA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and ...Nonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides. FmoA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and peptidyl carrier protein (PCP) domains. It uses α-methyl-l-serine to synthesize a 4-methyloxazoline ring, probably with another Cy domain in the preceding module FmoA2. Here, we determined the head-to-tail homodimeric structures of FmoA3 by X-ray crystallography (apo-form, with adenylyl-imidodiphosphate and α-methyl-l-seryl-AMP) and cryogenic electron microscopy single particle analysis, and performed site-directed mutagenesis experiments. The data revealed that α-methyl-l-serine can be accommodated in the active site because of the extra space around Ala688. The Cy domains of FmoA2 and FmoA3 catalyze peptide bond formation and heterocyclization, respectively. FmoA3's Cy domain seems to lose its donor PCP binding activity. The collective data support a proposed catalytic cycle of FmoA3.
History
DepositionJan 22, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 10, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 7, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jun 30, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title
Revision 1.3Nov 29, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Nonribosomal peptide synthetase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)126,30713
Polymers125,2441
Non-polymers1,06312
Water181
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, SEC-MALS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area880 Å2
ΔGint-19 kcal/mol
Surface area36970 Å2
MethodPISA
Unit cell
Length a, b, c (Å)164.206, 102.719, 137.412
Angle α, β, γ (deg.)90.000, 117.014, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Nonribosomal peptide synthetase / Nonribosomal peptide


Mass: 125243.695 Da / Num. of mol.: 1 / Mutation: S1046A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces sp. Sp080513GE-23 (bacteria)
Gene: fmoA3 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A077JG85

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Non-polymers , 5 types, 13 molecules

#2: Chemical ChemComp-AMP / ADENOSINE MONOPHOSPHATE / Adenosine monophosphate


Mass: 347.221 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H14N5O7P / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP*YM
#3: Chemical ChemComp-EW6 / alpha-methyl-L-serine / (2S)-2-azanyl-2-methyl-3-oxidanyl-propanoic acid


Type: L-peptide linking / Mass: 119.119 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H9NO3 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#5: Chemical
ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Cl
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 4.23 Å3/Da / Density % sol: 70.93 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7 / Details: 0.1M HEPES pH 7.0, 0.5M Li2SO4, 10% PEG 8000

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Data collection

DiffractionMean temperature: 95 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Photon Factory / Beamline: BL-1A / Wavelength: 1.1 Å
DetectorType: DECTRIS EIGER X 4M / Detector: PIXEL / Date: Nov 21, 2017
RadiationMonochromator: Si111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1 Å / Relative weight: 1
ReflectionResolution: 3.3→48.23 Å / Num. obs: 30791 / % possible obs: 100 % / Redundancy: 7.2 % / Biso Wilson estimate: 104.59 Å2 / CC1/2: 0.999 / Net I/σ(I): 14.5
Reflection shellResolution: 3.3→3.48 Å / Rmerge(I) obs: 1.011 / Num. unique obs: 4453 / CC1/2: 0.818

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Processing

Software
NameVersionClassification
PHENIX1.15.2_3472refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6LTA

6lta


Resolution: 3.3→48.23 Å / SU ML: 0.471 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 30.6125
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.267 1594 5.18 %
Rwork0.2194 29173 -
obs0.222 30767 99.91 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 97.92 Å2
Refinement stepCycle: LAST / Resolution: 3.3→48.23 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6525 0 56 1 6582
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01026743
X-RAY DIFFRACTIONf_angle_d1.29639261
X-RAY DIFFRACTIONf_chiral_restr0.06541050
X-RAY DIFFRACTIONf_plane_restr0.00921231
X-RAY DIFFRACTIONf_dihedral_angle_d8.55715282
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.3-3.410.39191350.33542609X-RAY DIFFRACTION99.96
3.41-3.530.33491590.26862634X-RAY DIFFRACTION99.75
3.53-3.670.28121150.24212643X-RAY DIFFRACTION99.82
3.67-3.840.30231200.22682677X-RAY DIFFRACTION100
3.84-4.040.29251470.22012656X-RAY DIFFRACTION100
4.04-4.290.25321480.20122640X-RAY DIFFRACTION100
4.29-4.620.23081590.18142649X-RAY DIFFRACTION100
4.62-5.090.20571480.17882649X-RAY DIFFRACTION100
5.09-5.820.32671440.22182642X-RAY DIFFRACTION100
5.82-7.330.24631540.23422675X-RAY DIFFRACTION99.96
7.33-48.230.25721650.22032699X-RAY DIFFRACTION99.48

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