The molybdenum cofactor sulfurase ABA3 from Arabidopsis thaliana is needed for post-translational activation of aldehyde oxidase and xanthine dehydrogenase by transferring a sulfur atom to the desulfo-molybdenum cofactor of these enzymes. ABA3 is a two-domain protein consisting of an NH(2)-terminal NifS-like cysteine desulfurase domain and a C-terminal domain of yet undescribed function. The NH(2)-terminal domain of ABA3 decomposes l-cysteine to yield elemental sulfur, which subsequently is bound as persulfide to a conserved protein cysteinyl residue within this domain. In vivo, activation of aldehyde oxidase and xanthine dehydrogenase also depends on the function of the C-terminal domain, as can be concluded from the A. thaliana aba3/sir3-3 mutant. sir3-3 plants are strongly reduced in aldehyde oxidase and xanthine dehydrogenase activities due to a substitution of arginine 723 by a lysine within the C-terminal domain of the ABA3 protein. Here we present first evidence for the function of the C-terminal domain and show that molybdenum cofactor is bound to this domain with high affinity. Furthermore, cyanide-treated ABA3 C terminus was shown to release thiocyanate, indicating that the molybdenum cofactor bound to the C-terminal domain is present in the sulfurated form. Co-incubation of partially active aldehyde oxidase and xanthine dehydrogenase with ABA3 C terminus carrying sulfurated molybdenum cofactor resulted in stimulation of aldehyde oxidase and xanthine dehydrogenase activity. The data of this work suggest that the C-terminal domain of ABA3 might act as a scaffold protein where prebound desulfo-molybdenum cofactor is converted into sulfurated cofactor prior to activation of aldehyde oxidase and xanthine dehydrogenase.
"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). "
[Show abstract][Hide abstract] ABSTRACT: Molybdenum cofactor sulfurases (MCSUs) are important enzymes for plant development and response to environmental
queues, including processes such as nitrogen metabolism and regulation of the abscisic acid levels in plant tissues. We cloned and
sequenced MCSU gene from barley and performed in silico comparison with rice, tomato, and Arabidopsis. Physico-chemical properties
and subcellular predictions were found to be similar in different plant species. All MCSUs had three critical domains: aminotransferase
class-V (Pfam: PF00266), MOSC N-terminal beta barrel (Pfam: PF03476), and MOSC (Pfam: PF03473). Secondary structure analysis
revealed that random coils were the most abundant, followed by α-helices and extended strands. Predicted binding sites of MCSUs
were different in barley and Arabidopsis, whereas rice and tomato showed the same pattern. A conserved triple-cysteine motif was
detected in all MCSUs with cys438-cys440-cys445, cys431-cys433-cys438, cys428-cys430-cys435, and cys425-cys427-cys432 in barley,
rice, Arabidopsis, and tomato, respectively. Furthermore, a 3D structure analysis indicated that structural divergences were present in
all MCSUs, even in the core domain structure. Phylogenetic analysis of MCSUs revealed that monocot–dicot divergence was clearly
observed with high bootstrap values. The results of this study will contribute to the understanding of MCSU genes and proteins in plants.
The data of this study will also constitute a scientific basis for wet-lab and in silico studies of MCSUs.
Turkish Journal of Agriculture and Forestry 03/2015; 39. DOI:10.3906/tar-1411-68 · 0.93 Impact Factor
"ABA3 is a two-domain protein with a N-terminal L-cysteine desulfurase domain that decomposes L-cysteine to L-alanine and sulfur (Bittner et al., 2001; Heidenreich et al., 2005). The sulfur is bound as a persulfide to a strictly conserved cysteine residue of the protein (ABA3-Cys430SH + S2- = > ABA3-Cys430S-SH) and transferred to the Moco-binding C-terminal domain (Wollers et al., 2008). On the C-terminal domain, the persulfide sulfur is transformed into a molybdenum-bound sulfido ligand by replacing an oxygen ligand [pterin-MoO2(OH) + S2- = > pterin-MoOS(OH) + O2-]. "
[Show abstract][Hide abstract] ABSTRACT: In the form of molybdate the transition metal molybdenum is essential for plants as it is required by a number of enzymes that catalyze key reactions in nitrogen assimilation, purine degradation, phytohormone synthesis, and sulfite detoxification. However, molybdate itself is biologically inactive and needs to be complexed by a specific organic pterin in order to serve as a permanently bound prosthetic group, the molybdenum cofactor, for the socalled molybdo-enyzmes. While the synthesis of molybdenum cofactor has been intensively studied, only little is known about the uptake of molybdate by the roots, its transport to the shoot and its allocation and storage within the cell. Yet, recent evidence indicates that intracellular molybdate levels are tightly controlled by molybdate transporters, in particular during plant development. Moreover, a tight connection between molybdenum and iron metabolisms is presumed because (i) uptake mechanisms for molybdate and iron affect each other, (ii) most molybdo-enzymes do also require iron-containing redox groups such as iron-sulfur clusters or heme, (iii) molybdenum metabolism has recruited mechanisms typical for iron-sulfur cluster synthesis, and (iv) both molybdenum cofactor synthesis and extramitochondrial iron-sulfur proteins involve the function of a specific mitochondrial ABC-type transporter.
"It has been suggested that a persulfide sulfur is transferred from the NifS-like domain of ABA3 to the Moco in AO of plants . More recent studies showed that the C-terminal MOSC domain is able to bind Moco in a 1 : 1 ratio with a dissociation constant of 0.55 ± 0.14 μM, implying that the C-terminus of ABA3 represents the functional homologue of XdhC in bacteria . In addition, it was shown that the Moco bound on ABA3 existed in its sulfurated form. "
[Show abstract][Hide abstract] ABSTRACT: Biogenesis of prokaryotic molybdoenzymes is a complex process with the final step representing the insertion of a matured molybdenum cofactor (Moco) into a folded apoenzyme. Usually, specific chaperones of the XdhC family are required for the maturation of molybdoenzymes of the xanthine oxidase family in bacteria. Enzymes of the xanthine oxidase family are characterized to contain an equatorial sulfur ligand at the molybdenum center of Moco. This sulfur ligand is inserted into Moco while bound to the XdhC-like protein and before its insertion into the target enzyme. In addition, enzymes of the xanthine oxidase family bind either the molybdopterin (Mo-MPT) form of Moco or the modified molybdopterin cytosine dinucleotide cofactor (MCD). In both cases, only the matured cofactor is inserted by a proofreading process of XdhC. The roles of these specific XdhC-like chaperones during the biogenesis of enzymes of the xanthine oxidase family in bacteria are described.
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