A tungsten-specific maturation pathway governs cofactor assembly of a CO2-reducing formate dehydrogenase in Methylorubrum extorquens.

Tungsten-dependent formate dehydrogenases catalyze the reversible interconversion of carbon dioxide and formate and play key roles in microbial redox metabolism, yet the molecular basis for selective tungsten cofactor assembly in aerobic bacteria remains poorly understood. Here, we define a dedicated tungsten-specific maturation pathway that governs cofactor assembly and delivery to the carbon dioxide-reducing formate dehydrogenase MeFDH1 in Methylorubrum extorquens AM1. Targeted genetic deletions identified moeA1, mobB, and fdhD as essential for MeFDH1 activity and tungsten incorporation. Protein-protein interaction and structural modeling analyses place MoeA1 at the center of this pathway, where a MoeA1-MobB platform mediates tungstate insertion into the pterin scaffold, followed by MobA-dependent guanylylation to generate W-bis-molybdopterin guanine dinucleotide and FdhD-catalyzed sulfuration and cofactor delivery to MeFDH1. Mutagenesis of residues within the predicted metal-binding pocket and metal competition experiments further indicate that the electrostatic environment of the MoeA1 biases cofactor assembly toward tungsten. Together, our findings establish a tungsten-selective cofactor assembly pathway in an aerobic bacterium and offer strategies for engineering tungsten-dependent redox enzymes for applications in microbial carbon fixation and biocatalysis.