The trace element molybdenum (Mo) is the catalytic component of important enzymes involved in global nitrogen, sulfur, and carbon metabolism in both prokaryotes and eukaryotes. With the exception of nitrogenase, Mo is complexed by a pterin compound thus forming the biologically active molybdenum cofactor (Moco) at the catalytic sites of molybdoenzymes. The physiological roles and biochemical functions of many molybdoenzymes have been characterized. However, our understanding of the occurrence and evolution of Mo utilization is limited. This article focuses on recent advances in comparative genomics of Mo utilization in the three domains of life. We begin with a brief introduction of Mo transport systems, the Moco biosynthesis pathway, the role of posttranslational modifications, and enzymes that utilize Mo. Then, we proceed to recent computational and comparative genomics studies of Mo utilization, including a discussion on novel Moco-binding proteins that contain the C-terminal domain of the Moco sulfurase and that are suggested to represent a new family of molybdoenzymes. As most molybdoenzymes need additional cofactors for their catalytic activity, we also discuss interactions between Mo metabolism and other trace elements and finish with an analysis of factors that may influence evolution of Mo utilization.

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...

Classical xanthinuria is a rare autosomal recessive metabolic disorder caused by variants in the XDH (type I) or MOCOS (type II) genes. Thirteen Israeli kindred (five Jewish and eight Arab) and two isolated cases from Germany were studied between the years 1997 and 2013. Four and a branch of a fifth of these families were previously described. Here, we reported the demographic, clinical, molecular and biochemical characterizations of the remaining cases. Seven out of 20 affected individuals (35%) presented with xanthinuria-related symptoms of varied severity. Among the 10 distinct variants identified, six were novel: c.449G>T (p.(Cys150Phe)), c.1434G>A (p.(Trp478*)), c.1871C>G (p.(Ser624*)) and c.913del (p.(Leu305fs*1)) in the XDH gene and c.1046C>T (p.(Thr349Ileu)) and c.1771C>T (p.(Pro591Ser)) in the MOCOS gene. Heterologous protein expression studies revealed that the p.Cys150Phe variant within the Fe/S-I cluster-binding site impairs XDH biogenesis, the p.Thr349Ileu variant in the NifS-like domain of MOCOS affects protein stability and cysteine desulfurase activity, while the p.Pro591Ser and a previously described p.Arg776Cys variant in the C-terminal domain affect Molybdenum cofactor binding. Based on the results of haplotype analyses and historical genealogy findings, the potential dispersion of the identified variants is discussed. As far as we are aware, this is the largest cohort of xanthinuria cases described so far, substantially expanding the repertoire of pathogenic variants, characterizing structurally and functionally essential amino acid residues in the XDH and MOCOS proteins and addressing the population genetic aspects of classical xanthinuria.