Genetic dissection of cyclic pyranopterin monophosphate biosynthesis in plant mitochondria.

Janneke Balk,Andrew E. Maclean,Inga Kruse,Lionel Hill

doi:10.1042/bcj20170559

Janneke Balk, Andrew E. Maclean + Show 2 more

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https://doi.org/10.1042/bcj20170559

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Abstract

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 Arabidopsis thaliana mutants for lines with decreased activities of cytosolic FeS enzymes and Moco enzymes. We identified a new mutant allele of ATM3 (ABC transporter of the mitochondria 3), 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 CNX2, cofactor of nitrate reductase and xanthine dehydrogenase 2, encoding GTP 3′,8-cyclase, the first step in Moco biosynthesis which is localized in the mitochondria. A single-nucleotide polymorphism in cnx2-2 leads to substitution of Arg88 with Gln in the N-terminal FeS cluster-binding motif. cnx2-2 plants are small and chlorotic, with severely decreased Moco enzyme activities, but they performed better than a cnx2-1 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 cnx2-1 and cnx2-2, and accumulated more than 10-fold in seedlings mutated in the downstream gene CNX5. Interestingly, atm3-1 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 CNX2 and suggest that ATM3 is indirectly required for cPMP synthesis.

Highlights

Iron–sulfur (FeS) clusters and molybdenum cofactor (Moco) are two covalently bound metal cofactors that mediate different types of redox reactions
The biosynthesis of Moco has been studied in bacteria, archaea and eukaryotes
Relatively little is known about the first step of the pathway carried out by CNX2 and CNX3, other than their localization in the mitochondrial matrix and a possible involvement of the ABC transporter of the mitochondria 3 (ATM3) exporter [9]

Summary

Introduction

Iron–sulfur (FeS) clusters and molybdenum cofactor (Moco) are two covalently bound metal cofactors that mediate different types of redox reactions. In the hope of identifying novel gene products acting upstream or downstream of ATM3, we selected two lines, xd22 and xd105, from the pool of uncharacterized sirtinol-resistant mutants based on (i) growth phenotypes found in atm3 alleles, such as chlorosis and narrow leaves [23] (Figure 2A); (ii) a strong decrease in AldOx activity (Figure 2B) and (iii) decreased cytosolic aconitase activity relative to the mitochondrial isozymes (Figure 2C).

Results

Conclusion