Dimerization of the plant molybdenum insertase Cnx1E is required for synthesis of the molybdenum cofactor

Abstract

The molybdenum cofactor (Moco) is a redox active prosthetic group, essentially required for numerous enzyme-catalyzed two electron transfer reactions. Moco is synthesized by an evolutionarily old and highly conserved multistep pathway. In the last step of Moco biosynthesis, the molybdenum center is inserted into the final Moco precursor adenylated molybdopterin (MPT-AMP). This unique and yet poorly characterized maturation reaction finally yields physiologically active Moco. In the model plant Arabidopsis, the two domain enzyme, Cnx1, is required for Moco formation. Recently, a genetic screen identified novel Arabidopsis cnx1 mutant plant lines each harboring a single amino acid exchange in the N-terminal Cnx1E domain. Biochemical characterization of the respective recombinant Cnx1E variants revealed two different amino acid exchanges (S197F and G175D) that impair Cnx1E dimerization, thus linking Cnx1E oligomerization to Cnx1 functionality. Analysis of the Cnx1E structure identified Cnx1E active site-bound molybdate and magnesium ions, which allowed to fine-map the Cnx1E MPT-AMP-binding site.

Insights into the Cnx1E catalyzed MPT-AMP hydrolysis

“…Within the Cnx1E active site, MPT-AMP adopts a conformation that is different from the one found in the Cnx1G MPT-AMP co-structure [11] and that properly orients the dithiolene motif toward enzyme bound molybdate [14,15]. Subsequently the MPT-AMP phosphoric anhydride bond is hydrolyzed, a reaction that is believed to be the prerequisite for enzyme catalyzed molybdate insertion into the MPT dithiolene motif [13][14][15]. Recent work suggested that the Cnx1E catalyzed molybdate insertion reaction involves the relocation of molybdate from the initial oxo-anion binding site to the insertion site [15].…”

“…Cnx1E variant D274E was generated following the QuikChange (Agilent Technologies) protocol modified for the use of Phusion ® High-Fidelity DNA Polymerase (Thermo Fischer). As described earlier [15], we used the Cnx1E wild-type pGPlus expression vector [14] as template for PCR-based mutagenesis. The sequence of the primer pair used to introduce the single amino acid exchange D274E was 5´-gggagacagggaGttcgtcaagccattactcgaag-3´and 5´-agtaatggcttgacgaaCtccctgtctcccattga-3´(mismatches in upper case).…”

“…In vivo interaction studies revealed the plant MPT synthase complex and the molybdenum insertase (Mo-insertase) to interact with each other, thus providing the framework necessary for efficient, protected and directed metabolite transfer [6]. The molybdate insertion reaction involves both functional domains of Mo-insertases, namely Eand G-domain [7] whose role for the Mo-insertion reaction has been studied in detail using the plant (Arabidopsis thaliana) Mo-insertase Cnx1 as model enzyme [8][9][10][11][12][13][14][15]. Notably, these domains are reactive as separately expressed domains (prokaryotes) or fused together (eukaryotes, except the lower alga Chlamydomonas reinhardtii [16]).…”

“…Subsequently, MPT-AMP is transferred to the Mo-insertase E-domain, which requires molybdate to be bound to the E-domain oxo-anion entry site [15]. Within the Cnx1E active site, MPT-AMP adopts a conformation that is different from the one found in the Cnx1G MPT-AMP co-structure [11] and that properly orients the dithiolene motif toward enzyme bound molybdate [14,15]. Subsequently the MPT-AMP phosphoric anhydride bond is hydrolyzed, a reaction that is believed to be the prerequisite for enzyme catalyzed molybdate insertion into the MPT dithiolene motif [13][14][15].…”

Aminopyrazine Pathway to the Moco Metabolite Dephospho Form A

“…[1] Next to Moco/MPT quantification of recombinant Moco dependent enzymes, [2,3] quantification of dephospho Form A( 4)h as also become of key importance for the determinationo ft he Moco/MPT content also of thoseproteins that are involved in the biosynthesis of Moco. [4,5] The detection limit of 4 by HPLC/fluorescence spectroscopy is in the picomolarr ange, which makes that quantification protocol suitable forb oth recombinant biochemistry andf or the characterization of biological samples, for example mutants trains. [6,7] The procedure was first reported by Rajagopalan and co-workers, [8] and modified later.…”