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- EMDB-34812: Cryo-EM structure of the bi-functional malonyl-CoA reductase from... -

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Basic information

Entry
Database: EMDB / ID: EMD-34812
TitleCryo-EM structure of the bi-functional malonyl-CoA reductase from Roseiflexus castenholzii
Map data
Sample
  • Complex: Malonyl-coenzyme A reductase/Malonate semialdehyde reductase/Short-chain dehydrogenase
    • Protein or peptide: Short-chain dehydrogenase/reductase SDR
KeywordsThe 3-hydroxypropionate cycle / Bifunctional enzyme / Short chain dehydrogenase / OXIDOREDUCTASE
Function / homologyoxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor / short 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)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 3.35 Å
AuthorsZhang X / Xu X / Xin J
Funding support China, 1 items
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, 2022-
Header (metadata) releaseMay 31, 2023-
Map releaseMay 31, 2023-
UpdateSep 20, 2023-
Current statusSep 20, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34812.png

Downloads & links

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Map

FileDownload / File: emd_34812.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-5.394344 - 7.578553
Average (Standard dev.)0.0056791375 (±0.18121238)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 214.00002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_34812_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Half map: #1

Fileemd_34812_half_map_2.map
Projections & Slices
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Sample components

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Entire : Malonyl-coenzyme A reductase/Malonate semialdehyde reductase/Shor...

EntireName: Malonyl-coenzyme A reductase/Malonate semialdehyde reductase/Short-chain dehydrogenase
Components
  • Complex: Malonyl-coenzyme A reductase/Malonate semialdehyde reductase/Short-chain dehydrogenase
    • Protein or peptide: Short-chain dehydrogenase/reductase SDR

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Supramolecule #1: Malonyl-coenzyme A reductase/Malonate semialdehyde reductase/Shor...

SupramoleculeName: Malonyl-coenzyme A reductase/Malonate semialdehyde reductase/Short-chain dehydrogenase
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The full-length MCR is a homodimer both in solution and cryo-EM structure.
Source (natural)Organism: Roseiflexus castenholzii DSM 13941 (bacteria)

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Macromolecule #1: Short-chain dehydrogenase/reductase SDR

MacromoleculeName: Short-chain dehydrogenase/reductase SDR / type: protein_or_peptide / ID: 1
Details: F269L is a natural mutation occurred during recombinant expression of the protein
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Roseiflexus castenholzii DSM 13941 (bacteria)
Molecular weightTheoretical: 134.268375 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSTVRRLEGK VALITGGAGN IGEVITRRFL AEGATVVITG RNAEKLAVYR RRLIDEERVA PERVVALRMD GSDIAQVRAG VAQIVHGGT DVPIPLHRID ILVNNAGSAG PRRRLVDIPL EPSEVQPPDS ETLAQAVGNL VGITWNLTRA AAPHMPSGSS V INISTIFS ...String:
MSTVRRLEGK VALITGGAGN IGEVITRRFL AEGATVVITG RNAEKLAVYR RRLIDEERVA PERVVALRMD GSDIAQVRAG VAQIVHGGT DVPIPLHRID ILVNNAGSAG PRRRLVDIPL EPSEVQPPDS ETLAQAVGNL VGITWNLTRA AAPHMPSGSS V INISTIFS RTDYYGRIAY VAPKAALNAL SDGLARELGV RGIRVNTIYP GPIESERIYT MFQAMDALKG QPEGDTASGF LR MMRLSRI DQNGEVVKRF PSPVDVANTA VLLASDESAA FTGHAFEVTH GMEVPTESRT TFVSRPGLRS VDATGKVILI CAG DQVDDA VALADTLRSC RATVVIGFRD PRALEKASVL LREPRHALAA DMYGRPTMTA EARLVRLDPL DPRAAAQTLE QIHA ELGAI HHAVVLPGQS RHAPSASLIE VDDQVVERFL HQELVGTIAL ARELARFWEE YPSGSSMHRV LFVSNPDDQQ GNQYS HILR AAVEQLVRVW RHESEYDSVN PAHQQEGQSS AAVWANQLIR YVNNEMANLD FTCAWVAKLL GSDRRIAEIN LYLPEE IVG TIGVHNPGFG WAESLFGLHM GKVALITGGS AGIGGQIGRL LALSGAHVML AARNADQLEQ MRASIVREVR DASYPDA ES RVAIFPGSDV SDIDGLERLV NHTVRVFGKV DYLINNAGIA GAEEMVIDMP VDAWRHTLRA NLISNYALLR RLAPQMKA A GGAYVLNVSS YFGGEKYVAI PYPNRSDYAV SKAGQRAMVE SLARFLGPEI QINAIAPGPV EGERLKGAGS RPGLFMRRA RLILENKRLN EVFAALLAAR HEGATIADLL PDLFANDIQS IANSAAMPAP LRRLATMLRE TSDAGGSAQS YLMNATIARK LLNRLENGG YITLHDRRAL TVEPPEPFFT EAQIEREAIK VRDGILGMLH LQRMPTEFDV ALATVFYLAD RNVTGETFHP S GGLRFERT VTEGELFGKP GQQRLERLKG SVVYLIGEHL RQHLVLLART FLDEIHVARV VLLTETTQAA TDLAAELSDY EA AGRFVVI PTCGDIEGGI DRAMAEYGRP GPVISTPFRP LPDRALSARN GDWSSVLTTA EFEELVEQQI THHFRVARKA GLI EGANVT LVTPPTSARS TSEEFALANF VKTTLHALTA TAGAESERTV PHVPVNQVDL TRRARSEEPR TPSEEEEELQ RFVN AVLLT SAPLPTPLES RYRARIYRGN AITV

UniProtKB: Short-chain dehydrogenase/reductase SDR

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.30 mg/mL
BufferpH: 8
StainingType: NEGATIVE / Material: Uranyl Acetate
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.3 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Gene Rcas_2929 from alaphafold
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1766605
FSC plot (resolution estimation)

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