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- PDB-5kf6: Structure of proline utilization A from Sinorhizobium meliloti co... -

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Basic information

Entry
Database: PDB / ID: 5kf6
TitleStructure of proline utilization A from Sinorhizobium meliloti complexed with L-tetrahydrofuroic acid and NAD+ in space group P21
ComponentsBifunctional protein PutA
KeywordsOXIDOREDUCTASE / FLAVOENZYME / ROSSMANN FOLD / ALDEHYDE DEHYDROGENASE / PROLINE CATABOLISM / SUBSTRATE CHANNELING / BIFUNCTIONAL ENZYME
Function / homology
Function and homology information


proline dehydrogenase / proline dehydrogenase activity / L-glutamate gamma-semialdehyde dehydrogenase / 1-pyrroline-5-carboxylate dehydrogenase activity / proline catabolic process to glutamate / proline biosynthetic process / cytoplasmic side of plasma membrane / DNA-binding transcription factor activity / nucleotide binding / DNA binding
Similarity search - Function
Proline dehydrogenase PutA, domain I / Proline utilization A proline dehydrogenase N-terminal domain / Proline utilization A proline dehydrogenase N-terminal domain / Delta-1-pyrroline-5-carboxylate dehydrogenase 3 / Proline dehydrogenase PutA, domain II / Proline dehydrogenase PutA, domain I/II / DNA-binding domain of Proline dehydrogenase / Bifunctional protein PutA / Proline dehydrogenase domain / Proline dehydrogenase ...Proline dehydrogenase PutA, domain I / Proline utilization A proline dehydrogenase N-terminal domain / Proline utilization A proline dehydrogenase N-terminal domain / Delta-1-pyrroline-5-carboxylate dehydrogenase 3 / Proline dehydrogenase PutA, domain II / Proline dehydrogenase PutA, domain I/II / DNA-binding domain of Proline dehydrogenase / Bifunctional protein PutA / Proline dehydrogenase domain / Proline dehydrogenase / : / FAD-linked oxidoreductase-like / Aldehyde dehydrogenase, cysteine active site / Aldehyde dehydrogenases cysteine active site. / Aldehyde dehydrogenase domain / Aldehyde dehydrogenase family / Aldehyde dehydrogenase, N-terminal / Aldehyde dehydrogenase, C-terminal / Aldehyde/histidinol dehydrogenase
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / NICOTINAMIDE-ADENINE-DINUCLEOTIDE / TRIETHYLENE GLYCOL / TETRAHYDROFURAN-2-CARBOXYLIC ACID / Bifunctional protein PutA
Similarity search - Component
Biological speciesSinorhizobium meliloti (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.7 Å
AuthorsTanner, J.J.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM065546 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM061068 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P30GM103335 United States
National Science Foundation (NSF, United States)DBI-1156692 United States
CitationJournal: J Biol Chem / Year: 2016
Title: Structures of Proline Utilization A (PutA) Reveal the Fold and Functions of the Aldehyde Dehydrogenase Superfamily Domain of Unknown Function.
Authors: Min Luo / Thameesha T Gamage / Benjamin W Arentson / Katherine N Schlasner / Donald F Becker / John J Tanner /
Abstract: Aldehyde dehydrogenases (ALDHs) catalyze the NAD(P)-dependent oxidation of aldehydes to carboxylic acids and are important for metabolism and detoxification. Although the ALDH superfamily fold is ...Aldehyde dehydrogenases (ALDHs) catalyze the NAD(P)-dependent oxidation of aldehydes to carboxylic acids and are important for metabolism and detoxification. Although the ALDH superfamily fold is well established, some ALDHs contain an uncharacterized domain of unknown function (DUF) near the C terminus of the polypeptide chain. Herein, we report the first structure of a protein containing the ALDH superfamily DUF. Proline utilization A from Sinorhizobium meliloti (SmPutA) is a 1233-residue bifunctional enzyme that contains the DUF in addition to proline dehydrogenase and l-glutamate-γ-semialdehyde dehydrogenase catalytic modules. Structures of SmPutA with a proline analog bound to the proline dehydrogenase site and NAD bound to the ALDH site were determined in two space groups at 1.7-1.9 Å resolution. The DUF consists of a Rossmann dinucleotide-binding fold fused to a three-stranded β-flap. The Rossmann domain resembles the classic ALDH superfamily NAD-binding domain, whereas the flap is strikingly similar to the ALDH superfamily dimerization domain. Paradoxically, neither structural element performs its implied function. Electron density maps show that NAD does not bind to the DUF Rossmann fold, and small-angle X-ray scattering reveals a novel dimer that has never been seen in the ALDH superfamily. The structure suggests that the DUF is an adapter domain that stabilizes the aldehyde substrate binding loop and seals the substrate-channeling tunnel via tertiary structural interactions that mimic the quaternary structural interactions found in non-DUF PutAs. Kinetic data for SmPutA indicate a substrate-channeling mechanism, in agreement with previous studies of other PutAs.
History
DepositionJun 12, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 5, 2016Provider: repository / Type: Initial release
Revision 1.1Oct 19, 2016Group: Database references
Revision 1.2Nov 23, 2016Group: Database references
Revision 1.3Sep 20, 2017Group: Author supporting evidence / Database references / Category: citation / pdbx_audit_support
Item: _citation.journal_id_CSD / _pdbx_audit_support.funding_organization
Revision 1.4Nov 1, 2017Group: Author supporting evidence / Category: pdbx_struct_assembly_auth_evidence
Revision 1.5Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.6Sep 27, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_symmetry

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Bifunctional protein PutA
B: Bifunctional protein PutA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)268,49819
Polymers263,9232
Non-polymers4,57417
Water23,1671286
1
A: Bifunctional protein PutA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)134,0028
Polymers131,9621
Non-polymers2,0407
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Bifunctional protein PutA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)134,49611
Polymers131,9621
Non-polymers2,53410
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11400 Å2
ΔGint-27 kcal/mol
Surface area79170 Å2
MethodPISA
Unit cell
Length a, b, c (Å)101.400, 102.332, 125.909
Angle α, β, γ (deg.)90.000, 106.530, 90.000
Int Tables number4
Space group name H-MP1211
DetailsTHE AUTHORS STATE THAT SAXS PERFORMED AT MULTIPLE PROTEIN CONCENTRATIONS SHOWS THAT BOTH MONOMER AND DIMER ARE OBSERVED IN SOLUTION.

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Bifunctional protein PutA


Mass: 131961.656 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sinorhizobium meliloti (strain SM11) (bacteria)
Strain: SM11 / Gene: putA, SM11_chr0102 / Plasmid: pNIC28-Bsa4 / Production host: Escherichia coli (E. coli) / References: UniProt: F7X6I3

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Non-polymers , 7 types, 1303 molecules

#2: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Comment: FAD*YM
#3: Chemical ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE


Mass: 663.425 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Comment: NAD*YM
#4: Chemical ChemComp-TFB / TETRAHYDROFURAN-2-CARBOXYLIC ACID


Mass: 116.115 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C5H8O3
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical
ChemComp-PGE / TRIETHYLENE GLYCOL


Mass: 150.173 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C6H14O4
#7: Chemical ChemComp-PG4 / TETRAETHYLENE GLYCOL


Mass: 194.226 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18O5 / Comment: precipitant*YM
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1286 / Source method: isolated from a natural source / Formula: H2O

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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: 2.37 Å3/Da / Density % sol: 48.16 %
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: 0.1 M Hepes at pH 7.5, 0.1 M ammonium sulfate, 0.1 M lithium sulfate monohydrate, 50 mM MgCl2, and 25% (w/v) PEG 3350

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 4.2.2 / Wavelength: 1 Å
DetectorType: RDI CMOS_8M / Detector: CMOS / Date: Mar 21, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.7→60.35 Å / Num. obs: 265575 / % possible obs: 98.3 % / Redundancy: 3.6 % / Biso Wilson estimate: 15.85 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.066 / Net I/σ(I): 13.3
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsDiffraction-ID% possible all
1.7-1.732.70.46190.3
9.31-60.353.20.029181.2

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Processing

Software
NameVersionClassification
Aimless0.2.17data scaling
PHENIX(1.10.1_2155)refinement
PDB_EXTRACT3.2data extraction
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1TJ1, 3HAZ

1tj1

3haz


Resolution: 1.7→51.376 Å / SU ML: 0.21 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 21.62
RfactorNum. reflection% reflection
Rfree0.2219 13382 5.06 %
Rwork0.1904 --
obs0.192 264376 97.81 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 75.13 Å2 / Biso mean: 21.5067 Å2 / Biso min: 1.17 Å2
Refinement stepCycle: final / Resolution: 1.7→51.376 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17771 0 293 1286 19350
Biso mean--22.63 24.4 -
Num. residues----2407
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00718448
X-RAY DIFFRACTIONf_angle_d0.89825130
X-RAY DIFFRACTIONf_chiral_restr0.0522950
X-RAY DIFFRACTIONf_plane_restr0.0063261
X-RAY DIFFRACTIONf_dihedral_angle_d16.0711302
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.7-1.71930.31573930.26047633802689
1.7193-1.73950.30054040.25127864826893
1.7395-1.76080.28014430.22138199864296
1.7608-1.78310.26094160.21438375879198
1.7831-1.80650.25844470.200484718918100
1.8065-1.83130.22994540.193585038957100
1.8313-1.85740.23024630.1985338996100
1.8574-1.88520.25544540.228384958949100
1.8852-1.91460.44114670.38998012847995
1.9146-1.9460.4144180.3727892831092
1.946-1.97960.24774680.20284268894100
1.9796-2.01560.22864510.180385609011100
2.0156-2.05430.2484480.20285458993100
2.0543-2.09630.28514620.26288092855496
2.0963-2.14180.21674560.177885428998100
2.1418-2.19170.22484300.171385648994100
2.1917-2.24650.28764390.268192863196
2.2465-2.30720.27274200.2548048846894
2.3072-2.37510.21344640.171885479011100
2.3751-2.45180.21284710.174985098980100
2.4518-2.53940.21124390.18018483892299
2.5394-2.64110.21534500.17685388988100
2.6411-2.76130.20265110.18248449896099
2.7613-2.90680.21534640.177785338997100
2.9068-3.08890.19854340.177585628996100
3.0889-3.32740.19994170.18118572898999
3.3274-3.66210.19924350.17178530896599
3.6621-4.19190.16734620.14928466892899
4.1919-5.28040.1544470.13388602904999
5.2804-51.39920.18054550.168257871294
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)
10.57010.02510.21530.12030.03320.2131-0.0305-0.0349-0.0060.02290.0298-0.0066-0.00790.00590.00140.12490.00780.01280.07510.00860.103-26.051265.963492.7245
20.1972-0.01560.1320.2494-0.05470.4670.0171-0.04520.01480.0465-0.00030.0020.03-0.111-0.01920.085-0.00190.00860.115-0.01630.09775.850631.080990.8582
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain A and not (resname NAD or resname FAD)A0
2X-RAY DIFFRACTION2chain B and not (resname NAD or resname FAD)B0

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