Phylogenomic survey of GTDB r232 expands the taxonomic and ecological range of alkylsuccinate synthase and distinguishes divergent AssA-like paralogs
- 1Institutional Research Office, Manila Central University
- 2Department of Biology, College of Arts and Sciences, Manila Central University
Abstract
Alkylsuccinate synthase (AssA / MasD) catalyses fumarate-dependent anaerobic alkane activation. Characterised enzymes are confined to a few cultured sulfate reducers, nitrate-reducers and the archaeon Archaeoglobus fulgidus, and each acts over a narrow carbon-number range. Sequence similarity alone does not separate AssA from its glycyl-radical paralogs. Screening GTDB r232 recovered 1,272 AssA-like MAGs across 28 phyla. The catalytic glycyl-radical core is invariant across the recovered sequences, so it cannot identify AssA. Discrimination rests instead on two clade-variable features: completeness of the assABCDE operon and retention of the (1-methylalkyl)succinate synthase (MASS) pocket. On these criteria the 1,272 MAGs yield only 92 canonical AssA sequences (Clade A1), 89 of them with an assignable host MAG. The rest fall into a paralog-like Clade A2 (n = 29), a divergent AssA-like Clade B (n = 251) and a pyruvate-formate-lyase-paralog comparator (Clade C, n = 900). An assA marker screen retaining all homology hits therefore over-recovers non-degraders more than several fold. Canonical AssA nonetheless extends well beyond the cultured Desulfobacterota into additional bacterial phyla with no prior record, most of them on the evidence of a single MAG, and includes largely uncultivated hosts such as SAR324 and Spirochaetota. Clades A2 and B carry no accessory subunits and match neither the MASS pocket nor the benzylsuccinate-synthase pocket. Neither clade contains a characterised enzyme, so their substrates cannot be assigned. Carriers derive predominantly from anoxic, hydrocarbon-bearing habitats, and gene-tree/species-tree discordance is consistent with horizontal mobility of assA. Operon and pocket criteria therefore provide a benchmark against misannotation of glycyl-radical enzymes in environmental datasets. The substrate of Clades A2 and B is the priority for biochemical work.
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BibTeX
@article{tan2026phylogenomic,
title = {{Phylogenomic survey of GTDB r232 expands the taxonomic and ecological range of alkylsuccinate synthase and distinguishes divergent AssA-like paralogs}},
author = {Boonfei Tan and Christian Zafra and Charmaine Ng},
journal = {Scientific Reports},
year = {2026},
month = sep,
doi = {10.1038/s41598-026-71283-x},
url = {https://doi.org/10.1038/s41598-026-71283-x},
note = {CC BY},
}
RIS
TY - JOUR
TI - Phylogenomic survey of GTDB r232 expands the taxonomic and ecological range of alkylsuccinate synthase and distinguishes divergent AssA-like paralogs
AU - Boonfei Tan
AU - Christian Zafra
AU - Charmaine Ng
PY - 2026
DA - 2026/09/01
JO - Scientific Reports
DO - 10.1038/s41598-026-71283-x
UR - https://doi.org/10.1038/s41598-026-71283-x
AB - Alkylsuccinate synthase (AssA / MasD) catalyses fumarate-dependent anaerobic alkane activation. Characterised enzymes are confined to a few cultured sulfate reducers, nitrate-reducers and the archaeon Archaeoglobus fulgidus, and each acts over a narrow carbon-number range. Sequence similarity alone does not separate AssA from its glycyl-radical paralogs. Screening GTDB r232 recovered 1,272 AssA-like MAGs across 28 phyla. The catalytic glycyl-radical core is invariant across the recovered sequences, so it cannot identify AssA. Discrimination rests instead on two clade-variable features: completeness of the assABCDE operon and retention of the (1-methylalkyl)succinate synthase (MASS) pocket. On these criteria the 1,272 MAGs yield only 92 canonical AssA sequences (Clade A1), 89 of them with an assignable host MAG. The rest fall into a paralog-like Clade A2 (n = 29), a divergent AssA-like Clade B (n = 251) and a pyruvate-formate-lyase-paralog comparator (Clade C, n = 900). An assA marker screen retaining all homology hits therefore over-recovers non-degraders more than several fold. Canonical AssA nonetheless extends well beyond the cultured Desulfobacterota into additional bacterial phyla with no prior record, most of them on the evidence of a single MAG, and includes largely uncultivated hosts such as SAR324 and Spirochaetota. Clades A2 and B carry no accessory subunits and match neither the MASS pocket nor the benzylsuccinate-synthase pocket. Neither clade contains a characterised enzyme, so their substrates cannot be assigned. Carriers derive predominantly from anoxic, hydrocarbon-bearing habitats, and gene-tree/species-tree discordance is consistent with horizontal mobility of assA. Operon and pocket criteria therefore provide a benchmark against misannotation of glycyl-radical enzymes in environmental datasets. The substrate of Clades A2 and B is the priority for biochemical work.
SN - 2045-2322
ER -