The molybdenum cofactor (Moco) forms part of the catalytic center in all eukaryotic molybdenum enzymes and is synthesized in a highly conserved pathway. Among eukaryotes, very little is known about the processes taking place subsequent to Moco biosynthesis, i.e. Moco transfer, allocation, and insertion into molybdenum enzymes. In the model plant Arabidopsis thaliana, we identified a novel protein family consisting of nine members that after recombinant expression are able to bind The molybdenum cofactor (Moco)2 is a prosthetic group highly conserved in all kingdoms of life and consists of a tricyclic pterin, referred to as molybdopterin or metal-binding pterin (MPT) and a molybdenum (Mo) atom covalently bound to the dithiolate moiety of MPT (1). Moco is required for the activity of all Mo-dependent enzymes with the exception of nitrogenase (2). Molybdenum enzymes (Mo-enzymes) are essential for a broad variety of metabolic processes such as nitrate assimilation and phytohormone synthesis in plants (3) and sulfur detoxification and purine catabolism in mammals (4).Synthesis of Moco proceeds in a highly conserved multistep pathway, involving at least six proteins named Cnx in plants (3). Much is known about the final step of Moco biosynthesis where one Mo atom is ligated to the MPT dithiolate function, which is catalyzed by the two-domain protein Cnx1 (5, 6): the C-terminal Cnx1-G domain activates MPT by adenylation, which is handed over to the N-terminal Cnx1-E domain where it is converted to Moco by inserting Mo into MPT under simultaneous cleavage of the pyrophosphate bond.After completion of biosynthesis, Moco has to be allocated and inserted into the apoMo-enzymes. In prokaryotes, a complex of proteins synthesizing the last steps of Moco biosynthesis donates the mature cofactor to apoenzymes assisted by enzyme-specific chaperones (7). In eukaryotes, however, no Mo-enzyme-specific chaperone has been found. As free Moco is extremely sensitive to oxidation it is also assumed that Moco occurs permanently protein-bound in the cell. Therefore, a cellular Moco distribution system should meet two demands: (i) it should bind Moco subsequent to its synthesis, and (ii) it should maintain a directed flow of Moco from the Moco donor Cnx1-E to the Mo-dependent enzymes. This is important to ensure the fast and efficient incorporation of Moco into apoMo-enzymes. In the alga Chlamydomonas reinhardtii a Moco carrier protein (MCP) was identified that was found to bind Moco and protect it against oxidation (8 -10). Without any denaturing procedure, subsequent transfer of Moco from MCP to apo-nitrate reductase (NR) from Neurospora crassa mutant nit-1 was possible (10), thus indicating that MCP-bound Moco was readily transferable. These properties of Chlamydomonas MCP make it a promising candidate for being part of a cellular Moco delivery system. It is, however, unknown whether MCP is also able to donate Moco to Mo-enzymes other than NR.Here we present the cloning and characterization of Mocobinding proteins (MoBP)
CitationIdentification of persulfide-binding and disulfide-forming cysteine residues in the NifS-like domain of the molybdenum cofactor sulfurase ABA3 by cysteine-scanning mutagenesis. 2012, 441 The molybdenum cofactor sulfurase ABA3 from Arabidopsis thaliana catalyzes the sulfuration of the molybdenum cofactor of aldehyde oxidase and xanthine oxidoreductase, which represents the final activation step of these enzymes. ABA3 consists of an N-terminal NifS-like domain that exhibits Lcysteine desulfurase activity, and a C-terminal domain that binds sulfurated molybdenum cofactor. The strictly conserved cysteine430 in the NifS-like domain binds a persulfide intermediate, which is abstracted from the substrate L-cysteine and finally needs to be transferred to the molybdenum cofactor of aldehyde oxidase and xanthine oxidoreductase. In addition to cysteine430, another eight cysteine residues are located in the NifS-like domain, with two of them being highly conserved among molybdenum cofactor sulfurase proteins and at the same time being in close proximity to cysteine430. By determination of the number of surface-exposed cysteine residues and the number of persulfidebinding cysteines in combination with the sequential substitution of each of the nine cysteines, a second persulfide-binding cysteine residue, cysteine206, was identified. Furthermore, the active-site cysteine430 was found to be located on top of a loop structure, formed by the two flanking cysteines428 and cysteine435, which are likely to form an intramolecular disulfide bridge. These findings are confirmed by a structural model of the NifS-like domain, which indicates that cysteine428 and cysteine435 are in disulfide bond distance and that a persulfide transfer from cysteine430 to cysteine206 is indeed possible.Key words: ABA3, Arabidopsis thaliana, Moco sulfurase, active site loop, persulfide, MOSC INTRODUCTIONMolybdenum enyzmes catalyze diverse key reactions in the global cycles of carbon, nitrogen, and sulfur [1,2]. With the exception of bacterial nitrogenase, all molybdenum enyzmes contain the socalled molybdenum cofactor (Moco) in which the molybdenum is coordinated by the dithiolene group of a molybdopterin backbone. According to the coordination chemistry of the molybdenum ligands, eukaryotic molybdenum enzymes were previously divided into two families: enzymes of the sulfite oxidase family are characterized by a Moco, whose molybdenum additionally ligates two oxo ligands and a protein-derived cysteinyl sulfur, while enzymes of the xanthine oxidase family bind a Moco, whose molybdenum ligates one oxo-ligand, a hydroxyl group, and a terminal sulfur. In higher eukaryotes, sulfite oxidase and nitrate reductase are members of the sulfite oxidase family, whereas aldehyde oxidase and xanthine oxidoreductase belong to the xanthine oxidase family of molybdenum enzymes. With regard to the terminal sulfur ligand, the enzymes of the xanthine oxidase family are unique in that this particular ligand needs to be delivered in a specific enzymatic reaction [3]. T...
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