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Mechanism of molybdate insertion into pterin-based molybdenum cofactors
Corinna Probst1, Jing Yang2, Joern Krausze1
1Institute of Plant Biology, Technische Universität Braunschweig, Braunschweig, Germany.
Researchers elucidated the final step in molybdenum cofactor (Moco) synthesis, identifying Moco-AMP as a key intermediate. This discovery reveals the mechanism of molybdate insertion into molybdopterin, crucial for Moco-dependent enzymes.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- The molybdenum cofactor (Moco) is essential for numerous enzymes involved in critical biological processes.
- Molybdopterin (MPT) is the bidentate ligand chelating molybdenum in Moco.
- The precise mechanism and structure of Moco prior to enzyme insertion remain largely unknown.
Purpose of the Study:
- To elucidate the final maturation step of Moco synthesis.
- To determine the structure and mechanism of molybdate insertion into MPT.
- To identify intermediates in the Moco biosynthesis pathway.
Main Methods:
- X-ray crystallography of the Arabidopsis thaliana Mo-insertase variant Cnx1E S269D D274S.
- X-ray absorption spectroscopy to determine the Moco coordination sphere.
- Biochemical assays using adenylated MPT (MPT-AMP) and molybdate as substrates.
Main Results:
- Adenylated Moco (Moco-AMP) was identified as an unexpected intermediate in the Moco synthesis pathway.
- The first coordination sphere geometry of Moco within the Cnx1E active site was revealed by X-ray absorption spectroscopy.
- A mechanism for molybdate insertion into MPT-AMP was deduced based on structural data.
Conclusions:
- The study reveals the final maturation step of Moco synthesis, involving Moco-AMP.
- A mechanism for molybdate insertion into MPT-AMP has been proposed.
- This mechanism is likely conserved across all eukaryotic Mo-insertases due to structural and sequence similarities.
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