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- PDB-9o8u: (1-methylalkyl)succinate synthase alpha-beta-gamma-delta complex ... -

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Basic information

Entry
Database: PDB / ID: 9o8u
Title(1-methylalkyl)succinate synthase alpha-beta-gamma-delta complex with bound fumarate
Components
  • (1-methyl alkyl succinate synthase subunit ...) x 3
  • MasB protein
KeywordsLYASE / glycyl radical enzyme / (1-methylalkyl)succinate synthase / X-succinate synthase / alkylsuccinate synthase / Hydrocarbon degradation / fumarate addition
Function / homology
Function and homology information


lyase activity / cytosol
Similarity search - Function
Benzylsuccinate synthase gamma subunit / Benzylsuccinate synthase gamma subunit superfamily / BssC/TutF protein / : / Formate C-acetyltransferase glycine radical, conserved site / Glycine radical domain signature. / Pyruvate formate lyase domain / Pyruvate formate lyase-like / Pyruvate formate-lyase domain profile. / Glycine radical ...Benzylsuccinate synthase gamma subunit / Benzylsuccinate synthase gamma subunit superfamily / BssC/TutF protein / : / Formate C-acetyltransferase glycine radical, conserved site / Glycine radical domain signature. / Pyruvate formate lyase domain / Pyruvate formate lyase-like / Pyruvate formate-lyase domain profile. / Glycine radical / Glycine radical domain / Glycine radical domain profile.
Similarity search - Domain/homology
2,3-DIHYDROXY-1,4-DITHIOBUTANE / : / FUMARIC ACID / IRON/SULFUR CLUSTER / MasB protein / 1-methyl alkyl succinate synthase subunit MasC / 1-methyl alkyl succinate synthase subunit MasD / 1-methyl alkyl succinate synthase subunit Mas E
Similarity search - Component
Biological speciesAzoarcus sp. HxN1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsAndorfer, M.C. / Drennan, C.L.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM126982 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM129882 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM145910 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Structural basis for anaerobic alkane activation by a multisubunit glycyl radical enzyme.
Authors: Mary C Andorfer / Talya S Levitz / Jian Liu / Ankush Chakraborty / Devin T King-Roberts / Delight Nweneka / Christa N Imrich / Catherine L Drennan /
Abstract: X-succinate synthases (XSSs) are glycyl radical enzymes (GREs) that catalyze the addition of hydrocarbons to fumarate via radical chemistry, thereby activating them for microbial metabolism. To date, ...X-succinate synthases (XSSs) are glycyl radical enzymes (GREs) that catalyze the addition of hydrocarbons to fumarate via radical chemistry, thereby activating them for microbial metabolism. To date, the only structurally characterized XSS is benzylsuccinate synthase (BSS), which functionalizes toluene. A distinct subclass of XSSs acts on saturated hydrocarbons, which possess much stronger C(sp)-H bonds than toluene, suggesting mechanistic and structural differences from BSS. Here, we use cryogenic electron microscopy to determine the structure of one such enzyme, (1-methylalkyl)succinate synthase (MASS) from strain HxN1, which functionalizes -alkanes (C6-C8). The structure reveals an asymmetric dimer in which both sides contain a catalytic α-subunit and accessory γ-subunit. One α-subunit also binds two additional subunits, β and δ. The β-subunit binds a [4Fe-4S] cluster and adopts a fold similar to BSSβ. The β-subunit appears to regulate the flexibility of the α-subunit to enable opening of the active site, affording the binding of -alkane substrates. The δ-subunit, which lacks homology to known GRE subunits, adopts a rubredoxin-like fold that binds a single Fe ion, an architecture not previously reported for GREs. MASSδ occupies the same region of the α-subunit as the activating enzyme (AE) and may regulate the conformational changes required for glycyl radical installation. Structural comparisons between MASS and BSS reveal differences in how fumarate is bound and show amino acid substitutions that could account for the binding of alkanes versus toluene. Together, this structure offers insight into anaerobic alkane activation via fumarate addition.
History
DepositionApr 16, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 23, 2025Provider: repository / Type: Initial release
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Revision 1.1Aug 20, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 1-methyl alkyl succinate synthase subunit MasD
B: MasB protein
C: 1-methyl alkyl succinate synthase subunit MasC
D: 1-methyl alkyl succinate synthase subunit Mas E
E: 1-methyl alkyl succinate synthase subunit MasD
F: 1-methyl alkyl succinate synthase subunit MasC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)230,77012
Polymers229,3896
Non-polymers1,3816
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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1-methyl alkyl succinate synthase subunit ... , 3 types, 5 molecules AECFD

#1: Protein 1-methyl alkyl succinate synthase subunit MasD / (1-methylalkyl)succinate synthase alpha chain


Mass: 97053.289 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Azoarcus sp. HxN1 (bacteria) / Gene: masD / Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3) / References: UniProt: A9J4K4
#3: Protein 1-methyl alkyl succinate synthase subunit MasC / (1-methylalkyl)succinate synthase gamma chain


Mass: 6894.553 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Azoarcus sp. HxN1 (bacteria) / Gene: masC / Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3) / References: UniProt: A9J4K2
#4: Protein 1-methyl alkyl succinate synthase subunit Mas E / (1-methylalkyl)succinate synthase delta chain


Mass: 8033.105 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Azoarcus sp. HxN1 (bacteria) / Gene: masE / Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3) / References: UniProt: A9J4K6

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Protein , 1 types, 1 molecules B

#2: Protein MasB protein / (1-methylalkyl)succinate synthase beta chain


Mass: 13460.091 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Azoarcus sp. HxN1 (bacteria) / Gene: masB / Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3) / References: UniProt: A9J4K0

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Non-polymers , 4 types, 6 molecules

#5: Chemical ChemComp-FUM / FUMARIC ACID


Mass: 116.072 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H4O4 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-DTT / 2,3-DIHYDROXY-1,4-DITHIOBUTANE / 1,4-DITHIOTHREITOL


Mass: 154.251 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O2S2 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationN

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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: Heterodimer complex of MASS alpha with beta, gamma, delta subunits
Type: COMPLEX
Details: one alpha subunits binds beta, gamma, delta subunits, and the other alpha subunit bind only gamma subunits.
Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.229 MDa / Experimental value: NO
Source (natural)Organism: Azoarcus sp. HxN1 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: NiCo21-DE3
Buffer solutionpH: 8
Details: 50 mM HEPES pH 8.0, 300 mM NaCl, 1 mM Fumarate, 1 mM DTT
Buffer component
IDConc.NameBuffer-ID
150 mMHEPES1
2300 mMSodium Chloride1
31 mMFumarate1
41 mMDTT1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 1.42 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
7UCSF ChimeraXmodel fitting
12RELION43D reconstruction
13PHENIXmodel refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 356678 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingSource name: AlphaFold / Type: in silico model

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