Phylogenomic survey of GTDB r232 expands the taxonomic and ecological range of alkylsuccinate synthase and distinguishes divergent AssA-like paralogs

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.