Mechanism of molybdate insertion into pterin-based molybdenum cofactors

The molybdenum cofactor (Moco) is found in the active site of numerous important enzymes that are critical to biological processes. The bidentate ligand that chelates molybdenum in Moco is the pyranopterin dithiolene (molybdopterin, MPT). However, neither the mechanism of molybdate insertion into MPT nor the structure of Moco prior to its insertion into pyranopterin molybdenum enzymes is known. Here, we report this final maturation step, where adenylated MPT (MPT–AMP) and molybdate are the substrates. X-ray crystallography of the Arabidopsis thaliana Mo-insertase variant Cnx1E S269D D274S identified adenylated Moco (Moco–AMP) as an unexpected intermediate in this reaction sequence. X-ray absorption spectroscopy revealed the first coordination sphere geometry of Moco trapped in the Cnx1E active site. We have used this structural information to deduce a mechanism for molybdate insertion into MPT–AMP. Given their high degree of structural and sequence similarity, we suggest that this mechanism is employed by all eukaryotic Mo-insertases. ; © The Author(s), under exclusive licence to Springer Nature Limited 2021. Received 29 April 2020. Accepted 27 April 2021. Published 28 June 2021. We thank A. Calean (TU Braunschweig) for excellent support with inductively coupled plasma mass spectrometry analysis. M.L.K. gratefully acknowledges support of this research by the National Institutes of Health (grant no. GM-057378). M.L.K. and J.Y. thank the UNM Center for Advanced Research Computing, supported in part by the National Science Foundation, for providing the computing resources used in this work. Work at Caltech was supported by the National Institutes of Health (NIH) grant no. GM045162. We acknowledge the Gordon and Betty Moore Foundation and the Beckman Institute at Caltech for their support of the Molecular Observatory at Caltech and the staff at beamlines 12-2 and 7-3. M.L.K. and J.Y. acknowledge the Stanford Synchrotron Radiation Lightsource, which is supported by the US Department of Energy, Office of ...