Genetic dissection of cyclic pyranopterin monophosphate biosynthesis in plant mitochondria.

Mitochondria play a key role in the biosynthesis of two metal cofactors, iron-sulfur (FeS) clusters and molybdenum cofactor (Moco). The two pathways intersect at several points, but a scarcity of mutants has hindered studies to better understand these links. We screened a collection of sirtinol-resistant mutants for lines with decreased activities of cytosolic FeS enzymes and Moco enzymes. We identified a new mutant allele of (), encoding the ATP-binding cassette transporter of the mitochondria 3 (systematic name ABCB25), confirming the previously reported role of ATM3 in both FeS cluster and Moco biosynthesis. We also identified a mutant allele in , , encoding GTP 3',8-cyclase, the first step in Moco biosynthesis which is localized in the mitochondria. A single-nucleotide polymorphism in leads to substitution of Arg88 with Gln in the N-terminal FeS cluster-binding motif. plants are small and chlorotic, with severely decreased Moco enzyme activities, but they performed better than a knockout mutant, which could only survive with ammonia as a nitrogen source. Measurement of cyclic pyranopterin monophosphate (cPMP) levels by LC-MS/MS showed that this Moco intermediate was below the limit of detection in both and , and accumulated more than 10-fold in seedlings mutated in the downstream gene Interestingly, mutants had less cPMP than wild type, correlating with previous reports of a similar decrease in nitrate reductase activity. Taken together, our data functionally characterize and suggest that ATM3 is indirectly required for cPMP synthesis.