Publications

• ABA3 is a molybdenum cofactor sulfurase required for activation of aldehyde oxidase and xanthine dehydrogenase in Arabidopsis thaliana.

  Bittner F., Oreb M., Mendel R.R.

  The xanthine oxidase class of molybdenum enzyzmes requires a terminal sulfur ligand at the active site. It has been proposed that a special sulfurase catalyzes the insertion of this ligand thereby activating the enzymes. Previous analyses of mutants in plants indicated that the genetic locus aba3 ... >> More

  The xanthine oxidase class of molybdenum enzyzmes requires a terminal sulfur ligand at the active site. It has been proposed that a special sulfurase catalyzes the insertion of this ligand thereby activating the enzymes. Previous analyses of mutants in plants indicated that the genetic locus aba3 is involved in this step leading to activation of the molybdenum enzymes aldehyde oxidase and xanthine dehydrogenase. Here we report the cloning of the aba3 gene from Arabidopsis thaliana and the biochemical characterization of the purified protein. ABA3 is a two-domain protein with a N-terminal NifS-like sulfurase domain and a C-terminal domain that might be involved in recognizing the target enzymes. Molecular analysis of three aba3 mutants identified mutations in both domains. ABA3 contains highly conserved binding motifs for pyridoxal phosphate and for a persulfide. The purified recombinant protein possesses a cysteine desulfurase activity, is yellow in color, and shows a NifS-like change in absorbance in the presence of L-cysteine. Pretreatment of ABA3 with a thiol-specific alkylating reagent inhibited its desulfurase activity. These data indicate a transsulfuration reaction similar to bacterial NifS. In a fully defined in vitro system, the purified protein was able to activate aldehyde oxidase by using L-cysteine as sulfur donor. Finally, we show that the expression of the aba3 gene is inducible by drought-stress. << Less

  J. Biol. Chem. 276:40381-40384(2001) [PubMed] [EuropePMC]

• Binding of sulfurated molybdenum cofactor to the C-terminal domain of ABA3 from Arabidopsis thaliana provides insight into the mechanism of molybdenum cofactor sulfuration.

  Wollers S., Heidenreich T., Zarepour M., Zachmann D., Kraft C., Zhao Y., Mendel R.R., Bittner F.

  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)-termina ... >> More

  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. << Less

  J. Biol. Chem. 283:9642-9650(2008) [PubMed] [EuropePMC]

• Drosophila melanogaster ma-l mutants are defective in the sulfuration of desulfo Mo hydroxylases.

  Wahl R.C., Warner C.K., Finnerty V., Rajagopalan K.V.

  Xanthine dehydrogenase was purified more than 1500-fold from crude extracts of wild type Drosophila melanogaster. Like the bovine milk and chicken liver enzymes, the purified Drosophila enzyme was inactivated by cyanide, and the cyanide-inactivated desulfo enzyme was reactivated by anaerobic incub ... >> More

  Xanthine dehydrogenase was purified more than 1500-fold from crude extracts of wild type Drosophila melanogaster. Like the bovine milk and chicken liver enzymes, the purified Drosophila enzyme was inactivated by cyanide, and the cyanide-inactivated desulfo enzyme was reactivated by anaerobic incubation with 1 mM sulfide and 1 mM dithionite. Application of the resulfuration procedure to crude extracts of Drosophila ma-l flies which slow pleiotropic deficiencies of xanthine dehydrogenase, aldehyde oxidase, and pyridoxal oxidase led to the emergence of xanthine dehydrogenase and aldehyde oxidase activities. Representatives of all the five known complementation groups of ma-l mutants were amenable to activation; 59-95% of wild type xanthine dehydrogenase activity and 1-7% of wild type aldehyde oxidase activity were reconstituted. Evidence for the identity of in vitro reconstituted xanthine dehydrogenase from ma-l mutants with wild type enzyme is presented. Since the inactive xanthine dehydrogenase and aldehyde oxidase proteins present in ma-l mutants are identical with the catalytically inactive desulfo forms obtained by cyanide treatment of active enzymes, these data constitute evidence for genetic control of the incorporation of the cyanolyzable sulfur of Mo hydroxylases. << Less

  J. Biol. Chem. 257:3958-3962(1982) [PubMed] [EuropePMC]

• Characterization of the NifS-like domain of ABA3 from Arabidopsis thaliana provides insight into the mechanism of molybdenum cofactor sulfuration.

  Heidenreich T., Wollers S., Mendel R.R., Bittner F.

  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 NH2-ter ... >> More

  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 NH2-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 post-translational activation of molybdenum enzymes rather than for iron-sulfur cluster assembly. << Less

  J. Biol. Chem. 280:4213-4218(2005) [PubMed] [EuropePMC]