Mining Bacterial Genomes for a ‘Hack-Proof’ Thiamin Biosynthesis Enzyme

Vitamins and their precursors are key currencies exchanged between within and between plants and their microbiomes. These vitamin transactions can be manipulated to favor or disfavor particular organisms. An example is the natural antibiotic bacimethrin, an analog of the thiamin precursor hydroxymethylpyrimidine (HMP) whose toxicity depends on replacing HMP in thiamin synthesis, i.e. on hacking into the thiamin pathway. Bacimethrin biosynthesis could in principle be installed in plants – but the plant platform would need first to be made bacimethrin-resistant (hack-proof). Bacimethrin enters the thiamin pathway via ThiD, a kinase present in plants and most bacteria that converts the biosynthetic intermediate HMP monophosphate to the diphosphate but also has a salvage activity that converts HMP (or bacimethrin) to the monophosphate. We therefore used comparative genomics to prospect for enzymes lacking the salvage activity. We uncovered a small protein of unknown function, often fused to the thiamin synthesis enzyme ThiE, that we predicted could replace the biosynthetic activity of ThiD but not its salvage activity. Genetic and biochemical analyses confirmed that this protein, named ThiD2, catalyzes the phosphorylation of HMP monophosphate, but not of HMP, bacimethrin, or other toxic HMP analogs. As a strictly monofunctional HMP monophosphate kinase, ThiD2 eliminates an inbuilt vulnerability of canonical ThiD.