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- PDB-8hi4: Cryo-EM structure of the bi-functional malonyl-CoA reductase from... -

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Basic information

Entry
Database: PDB / ID: 8hi4
TitleCryo-EM structure of the bi-functional malonyl-CoA reductase from Roseiflexus castenholzii
ComponentsShort-chain dehydrogenase/reductase SDR
KeywordsOXIDOREDUCTASE / The 3-hydroxypropionate cycle / Bifunctional enzyme / Short chain dehydrogenase
Function / homologyshort chain dehydrogenase / PKS_KR / Short-chain dehydrogenase/reductase SDR / NAD(P)-binding domain superfamily / nucleotide binding / Short-chain dehydrogenase/reductase SDR
Function and homology information
Biological speciesRoseiflexus castenholzii DSM 13941 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / cryo EM / Resolution: 3.35 Å
AuthorsZhang, X. / Xu, X. / Xin, J.
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
B: Short-chain dehydrogenase/reductase SDR


Theoretical massNumber of molelcules
Total (without water)268,5372
Polymers268,5372
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Short-chain dehydrogenase/reductase SDR


Mass: 134268.375 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: F269L is a natural mutation occurred during recombinant expression of the protein
Source: (gene. exp.) Roseiflexus castenholzii DSM 13941 (bacteria)
Gene: Rcas_2929 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A7NN59

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Malonyl-coenzyme A reductase/Malonate semialdehyde reductase/Short-chain dehydrogenase
Type: COMPLEX
Details: The full-length MCR is a homodimer both in solution and cryo-EM structure.
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Roseiflexus castenholzii DSM 13941 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: YES
EM stainingType: NEGATIVE / Material: Uranyl Acetate
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 1300 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refinedev_4746refinement
PHENIXdev_4746refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1766605 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 41.43 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00317823
ELECTRON MICROSCOPYf_angle_d0.63224289
ELECTRON MICROSCOPYf_dihedral_angle_d4.1492587
ELECTRON MICROSCOPYf_chiral_restr0.0422843
ELECTRON MICROSCOPYf_plane_restr0.0063221

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