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- PDB-8hi5: Crystal structure of the NADP+ and MSA bound C terminal domain of... -

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Basic information

Entry
Database: PDB / ID: 8hi5
TitleCrystal structure of the NADP+ and MSA bound C 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 / homology
Function and homology information


fatty acid elongation / oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor / nucleotide binding
Similarity search - Function
short chain dehydrogenase / PKS_KR / Short-chain dehydrogenase/reductase SDR / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
3-oxidanylidenepropanoic acid / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Short-chain dehydrogenase/reductase SDR
Similarity search - Component
Biological speciesRoseiflexus castenholzii DSM 13941 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 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)73,8563
Polymers73,0251
Non-polymers8312
Water1,45981
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: equilibrium centrifugation
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1150 Å2
ΔGint-3 kcal/mol
Surface area25320 Å2
Unit cell
Length a, b, c (Å)83.670, 83.670, 375.540
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number179
Space group name H-MP6522

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Components

#1: Protein Short-chain dehydrogenase/reductase SDR


Mass: 73024.969 Da / Num. of mol.: 1 / Fragment: C terminal domain
Source method: isolated from a genetically manipulated source
Details: The amino acid residues Met563-Ser566 are sequences derived from the expression vector, and His567-His572 are expression tag 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


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


Mass: 18.015 Da / Num. of mol.: 81 / 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: 2.6 Å3/Da / Density % sol: 52.67 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: 1.6 M sodium formate, and 0.1 M Bis-Tris propane pH 7.0

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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: 2.3→41.58 Å / Num. obs: 35934 / % possible obs: 99.92 % / Redundancy: 37.4 % / CC1/2: 0.999 / Net I/σ(I): 22.39
Reflection shellResolution: 2.3→2.382 Å / Num. unique obs: 3517 / CC1/2: 0.834

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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: 2.3→41.58 Å / SU ML: 0.33 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 26.6 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2491 1748 4.87 %
Rwork0.2111 --
obs0.2129 35922 99.95 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.3→41.58 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4946 0 54 81 5081
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0095095
X-RAY DIFFRACTIONf_angle_d1.0436928
X-RAY DIFFRACTIONf_dihedral_angle_d8.005727
X-RAY DIFFRACTIONf_chiral_restr0.054796
X-RAY DIFFRACTIONf_plane_restr0.017907
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.3-2.370.32731250.28512777X-RAY DIFFRACTION100
2.37-2.440.34471620.27652762X-RAY DIFFRACTION100
2.44-2.530.3241560.28072783X-RAY DIFFRACTION100
2.53-2.630.33521650.27632754X-RAY DIFFRACTION100
2.63-2.750.34641410.25752775X-RAY DIFFRACTION100
2.75-2.90.32991460.25832822X-RAY DIFFRACTION100
2.9-3.080.32651350.25062820X-RAY DIFFRACTION100
3.08-3.320.28541270.23862849X-RAY DIFFRACTION100
3.32-3.650.26621250.21922874X-RAY DIFFRACTION100
3.65-4.180.21091360.18572898X-RAY DIFFRACTION100
4.18-5.260.19951840.17422902X-RAY DIFFRACTION100
5.26-41.580.20351460.18193158X-RAY DIFFRACTION100

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