Binding of sulfurated molybdenum cofactor to the C-terminal domain of ABA3 from Arabidopsis thaliana provides insight into the mechanism of molybdenum cofactor sulfuration.
ABSTRACT 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.
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ABSTRACT: The biosynthesis of the molybdenum cofactors (Moco) is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum. Moco is the essential component of a group of redox enzymes, which are diverse in terms of their phylogenetic distribution and their architectures, both at the overall level and in their catalytic geometry. A wide variety of transformations are catalyzed by these enzymes at carbon, sulfur and nitrogen atoms, which include the transfer of an oxo group or two electrons to or from the substrate. More than 50 molybdoenzymes were identified to date. In all molybdoenzymes except nitrogenase, molybdenum is coordinated to a dithiolene group on the 6-alkyl side chain of a pterin called molybdopterin (MPT). The biosynthesis of Moco can be divided into three general steps, with a fourth one present only in bacteria and archaea: (1) formation of the cyclic pyranopterin monophosphate, (2) formation of MPT, (3) insertion of molybdenum into molybdopterin to form Moco, and (4) additional modification of Moco in bacteria with the attachment of a nucleotide to the phosphate group of MPT, forming the dinucleotide variant of Moco. This review will focus on the biosynthesis of Moco in bacteria, humans and plants.JBIC Journal of Biological Inorganic Chemistry 07/2014; · 3.16 Impact Factor
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ABSTRACT: Mo K-edge X-ray absorption spectroscopy has been used to probe as-isolated structures of the MOSC family proteins pmARC-1 and HMCS-CT. The Mo K-edge near-edge spectrum of HMCS-CT is shifted ∼2.5 eV to lower energy compared to the pmARC-1 spectrum, which indicates that as-isolated HMCS-CT is in a more reduced state than pmARC-1. Extended X-ray absorption fine structure analysis indicates significant structural differences between pmARC-1 and HMCS-CT, with the former being a dioxo site and the latter possessing only a single terminal oxo ligand. The number of terminal oxo donors is consistent with pmARC-1 being in the Mo VI oxidation state and HMCS-CT in the Mo IV state. These structures are in accord with oxygen-atom-transfer reactivity for pmARC-1 and persulfide bond cleavage chemistry for HMCS-CT. M embers of the molybdenum cofactor (Moco) sulfurase C-terminal (MOSC) domain superfamily of pyranopterin molybdenum (Mo) proteins are known to play significant roles in Moco maturation, prodrug activation, detoxification of possible mutagens, and the regulation of intracellular nitric oxide (NO) levels. 1−4 Moco sulfurase proteins are essential for the activation of Mo enzymes of the xanthine oxidase family. In this activation process, the N-terminal domain of Moco sulfurase decomposes free L-cysteine with concomitant release of sulfur, which is subsequently transferred to the Moco that is bound to the C-terminal domain of the protein. 5,6 Once sulfurated Moco is generated, the MOSC domain is capable of activating its target enzymes in a final maturation step. In addition to Moco sulfurases, the MOSC superfamily in eukaryotes also comprises the mitochondrial amidoxime reducing component (mARC) proteins, which are located in the outer mitochondrial membrane and catalyze the Moco-dependent reduction of a diverse range of N-hydroxylated substrates that include amidoximes, N-hydrox-ysulfonamides, and N-hydroxyguanidines. 4,7−12 Many of these substrates serve as prodrugs that are readily absorbed in the stomach and intestines and then are converted to their active forms by mARC enzymes. In spite of the role that mARC plays in prodrug activation, its exact biological function remains elusive. Yet, because of the known function of Moco sulfurases, a general role of MOSC proteins in metal−sulfur cluster biogenesis has been discussed. 13 Both mARC and Moco sulfurase have been shown to be related based on their sequence similarity 13,14 but have yet to be characterized by X-ray crystallography. This fundamental gap in the knowledge base has hindered our ability to determine key structure/function relationships for MOSC family proteins. Here, we use a combination of X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) at the Mo K-edge to probe the coordination environment of plant mARC isoform 1 (pmARC-1) and the human Moco sulfurase C-terminal domain (HMCS-CT). The XANES spectra for as-isolated, aerobic pmARC-1 and HMCS-CT are presented in Figure 1. These spectra derive fromInorganic Chemistry 08/2014; 53(18):9460. · 4.79 Impact Factor
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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.Frontiers in Plant Science 01/2014; 5:28. · 3.64 Impact Factor