The molybdenum cofactor is part of the active site of all molybdenum-dependent enzymes, except nitrogenase. The molybdenum cofactor consists of molybdopterin, a phosphorylated pyranopterin, with an ene-dithiolate coordinating molybdenum. The same pyranopterin-based cofactor is involved in metal coordination of the homologous tungsten-containing enzymes found in archea. The molybdenum cofactor is synthesized by a highly conserved biosynthetic pathway. In plants, the multidomain protein Cnx1 catalyses the insertion of molybdenum into molybdopterin. The Cnx1 G domain (Cnx1G), whose crystal structure has been determined in its apo form, binds molybdopterin with high affinity and participates in the catalysis of molybdenum insertion. Here we present two high-resolution crystal structures of Cnx1G in complex with molybdopterin and with adenylated molybdopterin (molybdopterin-AMP), a mechanistically important intermediate. Molybdopterin-AMP is the reaction product of Cnx1G and is subsequently processed in a magnesium-dependent reaction by the amino-terminal E domain of Cnx1 to yield active molybdenum cofactor. The unexpected identification of copper bound to the molybdopterin dithiolate sulphurs in both structures, coupled with the observed copper inhibition of Cnx1G activity, provides a molecular link between molybdenum and copper metabolism.

Nitrate assimilation is a key process for nitrogen (N) acquisition in green microalgae. Among Chlorophyte algae, Chlamydomonas reinhardtii has resulted to be a good model system to unravel important facts of this process, and has provided important insights for agriculturally relevant plants. In this work, the recent findings on nitrate transport, nitrate reduction and the regulation of nitrate assimilation are presented in this and several other algae. Latest data have shown nitric oxide (NO) as an important signal molecule in the transcriptional and posttranslational regulation of nitrate reductase and inorganic N transport. Participation of regulatory genes and proteins in positive and negative signaling of the pathway and the mechanisms involved in the regulation of nitrate assimilation, as well as those involved in Molybdenum cofactor synthesis required to nitrate assimilation, are critically reviewed.

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Molybdenum is an essential element for almost all living beings, which, in the form of a molybdopterin-cofactor, participates in the active site of enzymes involved in key reactions of carbon, nitrogen, and sulfur metabolism. This metal is taken up by cells in form of the oxyanion molybdate. Bacteria acquire molybdate by an ATP-bindingcassette (ABC) transport system in a widely studied process, but how eukaryotic cells take up molybdenum is unknown because molybdate transporters have not been identified so far. Here, we report a eukaryotic high-affinity molybdate transporter, encoded by the green alga Chlamydomonas reinhardtii gene MoT1. An antisense RNA strategy over the MoT1 gene showed that interference of the expression of this gene leads to the inhibition of molybdate transport activity and, in turn, of the Mo-containing enzyme nitrate reductase, indicating a function of MoT1 in molybdate transport. MOT1 functionality was also shown by heterologous expression in Saccharomyces cerevisiae. Molybdate uptake mediated by MOT1 showed a K m of Ϸ6 nM, which is the range of the lowest Km values reported and was activated in the presence of nitrate. Analysis of deduced sequence from the putative protein coded by MoT1 showed motifs specifically conserved in similar proteins present in the databases, and defines a family of membrane proteins in both eukaryotes and prokaryotes probably involved in molybdate transport and distantly related to plant sulfate transporters SULTR. These findings represent an important step in the understanding of molybdate transport, a crucial process in eukaryotic cells. micronutrient transport ͉ molybdenum ͉ molybdenum cofactor ͉ nitrate assimilation M olybdenum is one of the least abundant elements in organisms and is essential for molybdenum cofactor (Moco) biosynthesis. Moco is present in almost all living beings, taking part, as a prosthetic group, in the active site of key enzymes such as nitrate reductase, aldehyde oxidase, xanthine dehydrogenase, and sulfite oxidase. These enzymes participate in crucial processes for life such as nitrate assimilation, phytohormone biosynthesis, purine metabolism, and sulfite detoxification in plants, animals, and microorganisms (1, 2).Moco biosynthesis is a conserved pathway that consists of four main steps (3), conversion of GTP into cyclic pyranopterin monophosphate, synthesis of the molybdopterin dithiolate, adenylation of molybdopterin, and molybdenum-insertion reaction. In the fourth and final step, mature Moco is formed by the ligation of a single Mo atom to adenylated molybdopterin; this process directly depends on the intracellular availability of molybdenum. Deficiency in Moco biosynthesis results in a pleiotropic loss of all Mo-enzymes activity, which leads in humans to a severe metabolic disorder, and affected patients die in early childhood (4).In prokaryotes, molybdenum is taken up in an energydependent process by high-affinity molybdate transporters belonging to the ABC family (5). This system is formed by a periplasmic molybdate-bindin...

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