Identification of persulfide-binding and disulfide-forming cysteine residues in the NifS-like domain of the molybdenum cofactor sulfurase ABA3 by cysteine-scanning mutagenesis

Abstract

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...

The Molybdenum Cofactor

“…In a pyridoxal phosphate-dependent manner, the N-terminal domain of ABA3 decomposes L-cysteine to yield alanine and elemental sulfur (57), the latter being bound as a persulfide to a highly conserved cysteine residue of ABA3. The C-terminal domain of ABA3 shares a significant degree of similarity with the newly discovered mitochondrial amidoxime-reducing component proteins and was shown to bind sulfurated Moco, which receives the terminal sulfur via an intramolecular persulfide relay from the N-terminal domain (58,59). It is likely that subsequent to Moco sulfuration, ABA3 exchanges non-sulfurated for sulfurated Moco, thus activating its target molybdenum enzyme.…”

A Sulfurtransferase Is Essential for Activity of Formate Dehydrogenases in Escherichia coli

“…Determination of L-Cysteine Desulfurase Activity-L-Cysteine desulfurase activity was measured as described previously (21) by quantification of the produced L-alanine. L-Cysteine desulfurase activity is expressed in moles of alanine generated per min/mol of IscS.…”

Barley molybdenum cofactor sulfurase (MCSU): sequencing, modeling, and its comparison to other higher plants

“…Protein domains contain a stable 3D structure and are more conserved than sequences. Some changes are observed in protein folding during their evolution, even in the generation of new folding patterns (Lehrke et al, 2012). Structural divergences in MCSUs can be explained by some evolutionary genomic forces such as duplication, recombination, loss and transfer of a domain between species, and changes within the genomic (Rees and Howard, 2000).…”

“…The NifS domain functions as cysteine sulfurase using pyridoxal phosphate (PLP) as a cofactor. It transfers the sulfur from L-cysteine to target molecules and releases L-alanine (Heidenreich et al, 2005;Lehrke et al, 2012). The conserved C-terminal is considered to function in the recognition of molybdenum enzymes (Bittner et al, 2001).…”