Amidoximes can be used as prodrugs for amidines and related functional groups to enhance their intestinal absorption. These prodrugs are reduced to their active amidines. Other N-hydroxylated structures are mutagenic or responsible for toxic effects of drugs and are detoxified by reduction. In this study, a N-reductive enzyme system of pig liver mitochondria using benzamidoxime as a model substrate was identified. A protein fraction free from cytochrome b 5 and cytochrome b 5 reductase was purified, enhancing 250-fold the minor benzamidoxime-reductase activity catalyzed by the membrane-bound cytochrome b 5 /NADH cytochrome b 5 reductase system. This fraction contained a 35-kDa protein with homologies to the C-terminal domain of the human molybdenum cofactor sulfurase. Here it was demonstrated that this 35-kDa protein contains molybdenum cofactor and forms the hitherto ill defined third component of the N-reductive complex in the outer mitochondrial membrane. Thus, the 35-kDa protein represents a novel group of molybdenum proteins in eukaryotes as it forms the catalytic part of a three-component enzyme complex consisting of separate proteins. Supporting these findings, recombinant C-terminal domain of the human molybdenum cofactor sulfurase exhibited N-reductive activity in vitro, which was strictly dependent on molybdenum cofactor.
Assembly of iron-sulfur (Fe-S) clusters and maturation ofFe-S proteins in vivo require complex machineries. In Escherichia coli, under adverse stress conditions, this process is achieved by the SUF system that contains six proteins as follows: SufA, SufB, SufC, SufD, SufS, and SufE. Here, we provide a detailed characterization of the SufBCD complex whose function was so far unknown. Using biochemical and spectroscopic analyses, we demonstrate the following: (i) the complex as isolated exists mainly in a 1:2:1 (B:C:D) stoichiometry; (ii) the complex can assemble a [4Fe-4S] cluster in vitro and transfer it to target proteins; and (iii) the complex binds one molecule of flavin adenine nucleotide per SufBC 2 D complex, only in its reduced form (FADH 2 ), which has the ability to reduce ferric iron. These results suggest that the SufBC 2 D complex functions as a novel type of scaffold protein that assembles an Fe-S cluster through the mobilization of sulfur from the SufSE cysteine desulfurase and the FADH 2 -dependent reductive mobilization of iron.
The molybdenum cofactor sulfurase ABA3 from Arabidopsis thaliana specifically regulates the activity of the molybdenum enzymes aldehyde oxidase and xanthine dehydrogenase by converting their molybdenum cofactor from the desulfo-form into the sulfo-form. ABA3 is a two-domain protein with an NH 2 -terminal domain sharing significant similarities to NifS proteins that catalyze the decomposition of L-cysteine to L-alanine and elemental sulfur for iron-sulfur cluster synthesis. Although different in its physiological function, the mechanism of ABA3 for sulfur mobilization was found to be similar to NifS proteins. The protein binds a pyridoxal phosphate cofactor and a substrate-derived persulfide intermediate, and site-directed mutagenesis of strictly conserved binding sites for the cofactor and the persulfide demonstrated that they are essential for molybdenum cofactor sulfurase activity. In vitro, the NifS-like domain of ABA3 activates aldehyde oxidase and xanthine dehydrogenase in the absence of the C-terminal domain, but in vivo, the C-terminal domain is required for proper activation of both target enzymes. In addition to its cysteine desulfurase activity, ABA3-NifS also exhibits selenocysteine lyase activity. Although L-selenocysteine is unlikely to be a natural substrate for ABA3, it is decomposed more efficiently than L-cysteine. Besides mitochondrial AtNFS1 and plastidial AtNFS2, which are both proposed to be involved in iron-sulfur cluster formation, ABA3 is proposed to be a third and cytosolic NifS-like cysteine desulfurase in A. thaliana. However, the sulfur transferase activity of ABA3 is used for posttranslational activation of molybdenum enzymes rather than for iron-sulfur cluster assembly.NifS and NifS-like enzymes are present in almost all organisms and fulfill their main functions during iron-sulfur ([Fe-S]) cluster synthesis. Accordingly, they have a cysteine desulfurase activity that is required for the mobilization of sulfur from L-cysteine by simultaneous release of L-alanine. In all NifS-like enzymes, the sulfide is bound as a persulfide to a conserved cysteine residue of the protein, from which it is subsequently transferred to other target proteins such as the scaffold proteins NifU and/or IscU in bacteria to finally assemble [Fe-S] clusters. A pyridoxal phosphate (PLP) 1 cofactor bound to a conserved lysine residue is essential for this cysteine desulfurase activity (1). However, besides [Fe-S] cluster formation, other functions for NifS-like proteins are described as well. The Escherichia coli IscS protein also was found to be involved in the biosynthesis of thiamin and NAD ϩ and to be able to transfer a sulfur atom to uridine to produce a 4-thiouridine tRNA (2). In E. coli, another NifS-like protein, SufS (CsdB), is able to catalyze the elimination of selenium from L-selenocysteine more efficiently than the elimination of sulfur from L-cysteine (3). However, its physiological function is connected to its cysteine desulfurase activity, which is up to 50ϫ higher when SufS forms a complex with...
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