Functional characterization of a hyperthermophilic glycyl radical enzyme reveals a reductive glycerol pathway in archaea

The function of the glycyl radical enzyme (GRE) PFL2 from the hyperthermophilic archaeon Archaeoglobus fulgidus has remained unresolved since its discovery. Structural and sequence analyses initially suggested that PFL2 was a B12-independent glycerol dehydratase (GDHt), whereas subsequent structural and transcriptional evidence implicated it as a methylalkylsuccinate synthase involved in anaerobic alkane activation. Difficulties in reconstituting active PFL2 in vitro prevented either assignment from being established biochemically. Here, genome mining identified soluble activating enzymes that enabled us to demonstrate that PFL2 catalyzes glycerol dehydration to 3-hydroxypropionaldehyde (3-HPA). This reaction initiates the reductive glycerol pathway, in which 3-HPA is subsequently reduced to 1,3-propanediol (1,3-PD), an industrially important precursor to polymers and other materials. Although characterized in bacteria, this pathway has not previously been experimentally established in archaea. Motivated by this knowledge gap, we identified three oxidoreductases encoded in the A. fulgidus genome that catalyze the reduction of 3-HPA to 1,3-PD in vitro. Microscale thermophoresis revealed nanomolar-affinity binding between PFL2 and one of these oxidoreductases, AF0339 (apparent Kd = 70–90 nM), and AlphaFold-Multimer modeling predicted a continuous tunnel connecting their active sites, suggesting a possible route for intermediate transfer. Notably, PFL2 and AF0339 exhibited melting temperatures of 80.5 and 85.2 °C, respectively, revealing a thermally stable pair of enzymes catalyzing both steps of the pathway. Together, these findings establish PFL2 as an archaeal GDHt and provide experimental support for a reductive glycerol pathway in archaea. The thermostability of these enzymes further highlights their potential utility in biocatalytic 1,3-PD production.