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- PDB-8hi6: Crystal structure of the NADP+ and MSA bound N terminal domain of... -

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Basic information

Entry
Database: PDB / ID: 8hi6
TitleCrystal structure of the NADP+ and MSA bound N terminal domain of bi-functional malonyl-CoA reductase from Roseiflexus castenholzii
ComponentsShort-chain dehydrogenase/reductase SDR
KeywordsOXIDOREDUCTASE / The 3-hydroxypropionate cycle / Short chain dehydrogenase
Function / homologyshort chain dehydrogenase / PKS_KR / Short-chain dehydrogenase/reductase SDR / NAD(P)-binding domain superfamily / 3-oxidanylidenepropanoic acid / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Short-chain dehydrogenase/reductase SDR
Function and homology information
Biological speciesRoseiflexus castenholzii DSM 13941 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsZhang, X. / Wu, W.P. / Xu, X.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31870740, 32171227, 31570738 China
CitationJournal: mBio / Year: 2023
Title: Structural basis of a bi-functional malonyl-CoA reductase (MCR) from the photosynthetic green non-sulfur bacterium .
Authors: Xin Zhang / Jiyu Xin / Zhiguo Wang / Wenping Wu / Yutong Liu / Zhenzhen Min / Yueyong Xin / Bing Liu / Jun He / Xingwei Zhang / Xiaoling Xu /
Abstract: Malonyl-CoA reductase (MCR) is a NADPH-dependent bi-functional enzyme that performs alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities in the N- and C-terminal fragments, ...Malonyl-CoA reductase (MCR) is a NADPH-dependent bi-functional enzyme that performs alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities in the N- and C-terminal fragments, respectively. It catalyzes the two-step reduction of malonyl-CoA to 3-hydroxypropionate (3-HP), a key reaction in the autotrophic CO fixation cycles of green non-sulfur bacteria and the archaea . However, the structural basis underlying substrate selection, coordination, and the subsequent catalytic reactions of full-length MCR is largely unknown. For the first time, we here determined the structure of full-length MCR from the photosynthetic green non-sulfur bacterium (MCR) at 3.35 Å resolution. Furthermore, we determined the crystal structures of the N- and C-terminal fragments bound with reaction intermediates NADP and malonate semialdehyde (MSA) at 2.0 Å and 2.3 Å, respectively, and elucidated the catalytic mechanisms using a combination of molecular dynamics simulations and enzymatic analyses. Full-length MCR was a homodimer of two cross-interlocked subunits, each containing four tandemly arranged short-chain dehydrogenase/reductase (SDR) domains. Only the catalytic domains SDR1 and SDR3 incorporated additional secondary structures that changed with NADP-MSA binding. The substrate, malonyl-CoA, was immobilized in the substrate-binding pocket of SDR3 through coordination with Arg1164 and Arg799 of SDR4 and the extra domain, respectively. Malonyl-CoA was successively reduced through protonation by the Tyr743-Arg746 pair in SDR3 and the catalytic triad (Thr165-Tyr178-Lys182) in SDR1 after nucleophilic attack from NADPH hydrides. IMPORTANCE The bi-functional MCR catalyzes NADPH-dependent reduction of malonyl-CoA to 3-HP, an important metabolic intermediate and platform chemical, from biomass. The individual MCR-N and MCR-C fragments, which contain the alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities, respectively, have previously been structurally investigated and reconstructed into a malonyl-CoA pathway for the biosynthetic production of 3-HP. However, no structural information for full-length MCR has been available to illustrate the catalytic mechanism of this enzyme, which greatly limits our capacity to increase the 3-HP yield of recombinant strains. Here, we report the cryo-electron microscopy structure of full-length MCR for the first time and elucidate the mechanisms underlying substrate selection, coordination, and catalysis in the bi-functional MCR. These findings provide a structural and mechanistic basis for enzyme engineering and biosynthetic applications of the 3-HP carbon fixation pathways.
History
DepositionNov 18, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 31, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 28, 2023Group: 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.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Sep 13, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Sep 20, 2023Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Short-chain dehydrogenase/reductase SDR
hetero molecules


Theoretical massNumber of molelcules
Total (without water)64,2783
Polymers63,4471
Non-polymers8312
Water4,071226
1
A: Short-chain dehydrogenase/reductase SDR
hetero molecules

A: Short-chain dehydrogenase/reductase SDR
hetero molecules


Theoretical massNumber of molelcules
Total (without water)128,5576
Polymers126,8942
Non-polymers1,6634
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation10_665-y+1,-x+1,-z+1/61
Buried area6030 Å2
ΔGint-17 kcal/mol
Surface area38900 Å2
MethodPISA
Unit cell
Length a, b, c (Å)98.190, 98.190, 333.540
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number179
Space group name H-MP6522

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Components

#1: Protein Short-chain dehydrogenase/reductase SDR


Mass: 63446.832 Da / Num. of mol.: 1 / Fragment: N terminal domain
Source method: isolated from a genetically manipulated source
Details: His573-His578 are expression tag used for purification of the target protein
Source: (gene. exp.) Roseiflexus castenholzii DSM 13941 (bacteria)
Strain: DSM 13941 / HLO8 / Gene: Rcas_2929 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A7NN59
#2: Chemical ChemComp-FK2 / 3-oxidanylidenepropanoic acid


Mass: 88.062 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H4O3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-NAP / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE / Nicotinamide adenine dinucleotide phosphate


Mass: 743.405 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H28N7O17P3 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 226 / 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

CrystalDensity Matthews: 3.66 Å3/Da / Density % sol: 66.37 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: 1 M sodium malonate pH 5.0, 0.1 M sodium acetate trihydrate pH 4.5, and 3% PEG 20,000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL10U2 / Wavelength: 0.97915 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Oct 30, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97915 Å / Relative weight: 1
ReflectionResolution: 2→44.91 Å / Num. obs: 65288 / % possible obs: 99.92 % / Redundancy: 38 % / CC1/2: 0.999 / Net I/σ(I): 19.95
Reflection shellResolution: 2→2.03 Å / Num. unique obs: 2754 / CC1/2: 0.813

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
PDB_EXTRACT3.27data extraction
XDSdata reduction
XDSdata scaling
PHASERphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 8HI4

8hi4


Resolution: 2→44.91 Å / SU ML: 0.18 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 20.08 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2071 3256 4.99 %
Rwork0.1932 62033 -
obs0.1939 65288 99.92 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 80.5 Å2 / Biso mean: 32.1632 Å2 / Biso min: 16.52 Å2
Refinement stepCycle: final / Resolution: 2→44.91 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4065 0 79 226 4370
Biso mean--20.58 36.03 -
Num. residues----534
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 23

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2-2.030.29821250.252326292754100
2.03-2.060.28611440.239526242768100
2.06-2.10.23821410.222326612802100
2.1-2.130.25951440.218926352779100
2.13-2.170.21441350.213126392774100
2.17-2.210.22711480.210626232771100
2.21-2.260.21561170.214626722789100
2.26-2.310.24721240.21726612785100
2.31-2.360.23731530.212426752828100
2.36-2.420.23981360.208626492785100
2.42-2.480.24231460.207226512797100
2.48-2.560.24461470.193326612808100
2.56-2.640.24471610.204826652826100
2.64-2.730.22181500.206826852835100
2.73-2.840.22351400.199926702810100
2.84-2.970.24141290.209527292858100
2.97-3.130.24421450.210126862831100
3.13-3.330.24971350.200427122847100
3.33-3.580.21111360.193827422878100
3.58-3.940.19671400.173727612901100
3.94-4.510.15381610.157527582919100
4.51-5.680.15811560.169428142970100
5.68-44.910.1621430.18513031317499

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