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- PDB-7uch: AprA Methyltransferase 1 - GNAT in complex with Mn2+ , SAM, and D... -

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Basic information

Entry
Database: PDB / ID: 7uch
TitleAprA Methyltransferase 1 - GNAT in complex with Mn2+ , SAM, and Di-methyl-malonate
ComponentsAprA Methyltransferase 1
KeywordsTRANSFERASE / polyketide synthase
Function / homology
Function and homology information


phosphopantetheine binding / transferase activity
Similarity search - Function
Methyltransferase type 12 / Methyltransferase domain / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / Prokaryotic membrane lipoprotein lipid attachment site profile. / Winged helix-like DNA-binding domain superfamily / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
: / dimethylpropanedioic acid / S-ADENOSYL-L-HOMOCYSTEINE / Carrier domain-containing protein
Similarity search - Component
Biological speciesMoorena bouillonii (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.18 Å
AuthorsSkiba, M.A. / Lao, Y. / Smith, J.L.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)DK042303 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA108874 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)GM008353 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)GM008270 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)GM067550 United States
Citation
Journal: Acs Chem.Biol. / Year: 2022
Title: Structural Basis for Control of Methylation Extent in Polyketide Synthase Metal-Dependent C -Methyltransferases.
Authors: Lao, Y. / Skiba, M.A. / Chun, S.W. / Narayan, A.R.H. / Smith, J.L.
#1: Journal: ACS Chem Biol / Year: 2018
Title: Biosynthesis of t-Butyl in Apratoxin A: Functional Analysis and Architecture of a PKS Loading Module.
Authors: Meredith A Skiba / Andrew P Sikkema / Nathan A Moss / Andrew N Lowell / Min Su / Rebecca M Sturgis / Lena Gerwick / William H Gerwick / David H Sherman / Janet L Smith /
Abstract: The unusual feature of a t-butyl group is found in several marine-derived natural products including apratoxin A, a Sec61 inhibitor produced by the cyanobacterium Moorea bouillonii PNG 5-198. Here, ...The unusual feature of a t-butyl group is found in several marine-derived natural products including apratoxin A, a Sec61 inhibitor produced by the cyanobacterium Moorea bouillonii PNG 5-198. Here, we determine that the apratoxin A t-butyl group is formed as a pivaloyl acyl carrier protein (ACP) by AprA, the polyketide synthase (PKS) loading module of the apratoxin A biosynthetic pathway. AprA contains an inactive "pseudo" GCN5-related N-acetyltransferase domain (ΨGNAT) flanked by two methyltransferase domains (MT1 and MT2) that differ distinctly in sequence. Structural, biochemical, and precursor incorporation studies reveal that MT2 catalyzes unusually coupled decarboxylation and methylation reactions to transform dimethylmalonyl-ACP, the product of MT1, to pivaloyl-ACP. Further, pivaloyl-ACP synthesis is primed by the fatty acid synthase malonyl acyltransferase (FabD), which compensates for the ΨGNAT and provides the initial acyl-transfer step to form AprA malonyl-ACP. Additionally, images of AprA from negative stain electron microscopy reveal multiple conformations that may facilitate the individual catalytic steps of the multienzyme module.
History
DepositionMar 16, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 1, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 31, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _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: AprA Methyltransferase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,4895
Polymers74,8261
Non-polymers6644
Water5,747319
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)60.572, 88.283, 135.982
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

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Protein , 1 types, 1 molecules A

#1: Protein AprA Methyltransferase 1


Mass: 74825.836 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Moorena bouillonii (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A1U7N2Z8

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Non-polymers , 5 types, 323 molecules

#2: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-MU6 / dimethylpropanedioic acid


Mass: 132.115 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H8O4
#5: Chemical ChemComp-SAH / S-ADENOSYL-L-HOMOCYSTEINE / S-Adenosyl-L-homocysteine


Type: L-peptide linking / Mass: 384.411 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H20N6O5S / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 319 / 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: 2.43 Å3/Da / Density % sol: 49.37 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 11mg/mL protein in 50mM Tris pH7.4, 50mM NaCl, 10%(V/V) glycerol, 1mM S-adenosyl-L-homocysteine (SAH), and 5mM MnCl2. Well solution: 15% PEG 3350, 0.1M di-methyl-malonate Protein to well solution ratio is 2:2

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-B / Wavelength: 1.033 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 9, 2017
RadiationMonochromator: M / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.033 Å / Relative weight: 1
ReflectionResolution: 2.18→45.23 Å / Num. obs: 38749 / % possible obs: 99.87 % / Redundancy: 10.6 % / Biso Wilson estimate: 33.02 Å2 / CC1/2: 0.995 / CC star: 0.999 / Net I/σ(I): 7.37
Reflection shellResolution: 2.181→2.259 Å / Mean I/σ(I) obs: 1.18 / Num. unique obs: 3805 / CC1/2: 0.495 / CC star: 0.814 / % possible all: 99.42

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
PHASERphasing
XDSdata scaling
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6B3A

6b3a


Resolution: 2.18→45.23 Å / SU ML: 0.3282 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 26.5831
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2411 3656 5 %
Rwork0.193 69451 -
obs0.1955 38749 99.87 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 40.78 Å2
Refinement stepCycle: LAST / Resolution: 2.18→45.23 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5149 0 42 319 5510
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00755332
X-RAY DIFFRACTIONf_angle_d0.89977229
X-RAY DIFFRACTIONf_chiral_restr0.0522817
X-RAY DIFFRACTIONf_plane_restr0.0074923
X-RAY DIFFRACTIONf_dihedral_angle_d11.80111995
LS refinement shellResolution: 2.18→2.21 Å
RfactorNum. reflection% reflection
Rfree0.3526 135 -
Rwork0.3161 2622 -
obs--98.11 %

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