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Reconstitution of Molybdoenzymes with Bis-Molybdopterin Guanine Dinucleotide Cofactors

Abstract : Molybdoenzymes are ubiquitous, and play important roles in all kingdoms of life. The enzymes' cofactors comprise the metal molybdenum, a special organic ligand system called molybdopterin (MPT), additional small ligands like water, hydroxide, oxo-, sulfido-or selenido-functions and, in some enzymes, a coordination to the peptide chain of the protein via an amino acid ligand (e.g. serine, aspartate, cysteine or selenosysteine). The so-called molybdenum cofactor (Moco) is deeply buried in the protein at the end of a narrow funnel giving access only to the substrate. In 1974 an assay was developed by Nason and coworkers using the pleotrophic Neurospora crassa mutant nit-1 for the reconstitution of molybdoenzyme activities from crude extracts. These studies lead to the understanding that Moco is the common element in all molybdoenzymes from different organisms. The assay has been further developed since using specific molybdenum enzymes as source of Moco for the reconstitution of diverse purified apo-molybdoenzymes. Alternatively, the molybdenum cofactor can be synthesized in vitro from stable intermediates and can be inserted into apo-molybdoenzymes by the aid of specific Moco-binding chaperones. A general working protocol is described here for the insertion of the bis-molybdopterin guanine dunucleotide cofactor (bis-MGD) into its target molybdoenzyme using the example of Escherichia coli TMAO reductase. 2
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Paul Kaufmann, Chantal Iobbi-Nivol, Silke Leimkuhler. Reconstitution of Molybdoenzymes with Bis-Molybdopterin Guanine Dinucleotide Cofactors. Metalloproteins, pp.141-152, 2019, Methods in Molecular Biology, 978-1-4939-8863-1. ⟨hal-01927909⟩

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