Molybdenum in Biology - An Essential Trace Element
Protein binding
Comparative analysis of the molybdate transport proteins in various bacteria and archaea is reviewed. In both bacteria and archaea, molybdate is transported by an ABC-type transporter comprising three proteins, ModA (periplasmic binding protein), ModB (membrane protein) and ModC, the ATPase. The modABC operon expression is controlled by ModE-Mo. In the absence of the high-affinity molybdate transporter, molybdate is also transported by another ABC transporter which transports sulfate/thiosulfate as well as by a nonspecific anion transporter.
Self, W.T., Grunden, A. M., Hasona, A., and Shanmugam, K. T., Molybdate transport, Research in Microbiology, 2001, 152, 311-321.
To examine the biochemical mechanism by which heat-shock-protein, hsp90, exerts its essential positive function on certain signal transduction proteins, the effects of molybdate and geldanamycin on hsp90, function and structure were characterised. Molybdate inhibited hsp90-mediated p56(lck) biogenesis in the rabbit and firefly luciferase renaturation. Molybdate also reduced the amount of free hsp90 present in cell lysates, inhibited hsp90's ability to bind geldanamycin, and induced resistance to proteolysis at a specific region within the C-terminal domain of hsp90. In contrast, the hsp90 inhibitor geldanamycin prevented hsp90 from assuming natural or molybdate-induced conformations that allow salt-stable interactions with substrates. A specific region within the C-terminal domain of hsp90 (near residue 600) determines the mode by which hsp90 interacts with substrates and that the ability of hsp90 to cycle between alternative modes of interaction is obligatory for hsp90 function.
Hartson , S.D. , Thulasiraman, V., Huang, W.J., Whitesell, L., Matts, R.L. ,Molybdate inhibits hsp90, induces structural changes in its C-terminal domain, and alters its interactions with substrates, Biochemistry , 1999, 38 , 12, 3837-3849
Transport of molybdenum into bacteria involves a high-affinity ABC transporter system whose expression is controlled by a repressor protein called ModE. While molybdate transport is tightly coupled to utilization in some bacteria, other organisms have molybdenum storage proteins. One class of putative molybdate storage proteins is characterized by a sequence consisting of about 70 amino acids (Mop). A tandem repeat of Mop sequences also constitutes the molybdate binding domain of ModE. Results: The crystal structure of the 7 kDa Mop protein from the methanol-utilizing anaerobic eubacterium Sporomusa ovata grown in the presence of molybdate and tungstate has been determined. The protein occurs as highly symmetric hexamers binding eight oxoanions. Each peptide assumes a so- called OB fold, which has previously also been observed in ModE. There are two types of oxoanion binding sites in Mo at the interface between two or three peptides. All oxoanion binding sites were found to be occupied by WO4 rather than MoO 4. The biological function of proteins containing only Mop sequences is unknown, but they have been implicated in molybdate homeostasis and molybdopterin cofactor biosynthesis. While there are few indications that the S. ovata Mop binds pterin, the structure suggests that only the type-1 oxoanion binding sites would be sufficiently accessible to bind a cofactor. The observed occupation of the oxoanion binding sites by WO4 indicates that Mop might also be involved in controlling intracellular tungstate levels
Wagner, U.G., Stupperich, E., and Kratky, C., Structure of the molybdate/tungstate binding protein Mop front Sporomusa ovata, Structure, 2000, 8, 1127-1136.
In the blood, molybdenum binds to a-2-macroglobulins in the form of molybdate. Binding of molybdenum to the protein, spectrin also occurs on the erythrocyte membrane [Barceloux, 1999].
Barceloux, D.G., Molybdenum, Journal Of Toxicology-Clinical Toxicology , 1999, 37, 231-237.
A novel tungstate and molybdate binding protein has been discovered from the hyperthermophilic archaeon Pyrococcus furiosus. Its structural gene is present in the genome of numerous archaea and some bacteria. Using isothermal titration calorimetry, WtpA was observed to bind tungstate (dissociation constant [K-D] of 17 ± 7 pM) and molybdate (K-D of 11 ± 5 nM) with a stoichiometry of 1.0 mol oxoanion per mole of protein. A displacement titration of molybdate-saturated WtpA with tungstate showed that the tungstate effectively replaced the molybdate in the binding site of the protein.
Bevers, L. E., Hagedoorn, P. L., Krijger, G. C., and Hagen, W. R., Tungsten transport protein A (WtpA) in Pyrococcus futiosus: the first member of a new class of tungstate and molybdate transporters, Journal of Bacteriology, 2006, 188, 6498-6505.
Enzyme-specific proteins exist for the biogenesis of molybdoenzymes, coordinating Moco binding and insertion into their respective target proteins. So far, the requirement of such proteins for molybdoenzyme maturation has been described only for prokaryotes.
Neumann, M., Schulte, M., Junemann, N., Stocklein, W., and Leimkuhler, S., Rhodobacter capsulatus XdhC is involved in molybdenum cofactor binding and insertion into xanthine dehydrogenase, Journal of Biological Chemistry, 2006, 281, 15701-15708.
The molybdenum cofactor (Moco) is synthesized by an ancient and conserved biosynthetic pathway. In plants, the two-domain protein Cnx1 catalyzes the insertion of molybdenum into molybdopterin (MPT), a metal-free phosphorylated pyranopterin carrying an ene-dithiolate. A novel biosynthetic intermediate, adenylated molybdopterin, MPT-AMP has been ideintified. MPT-AMP and molybdate bind in an equimolar and cooperative way to the other N-terminal E domain (Cnx1E). Tungstate and sulfate compete for molybdate, which demonstrates the presence of an anion-binding site for molybdate. Cnx1E catalyzes the Zn2+-/Mg2+-dependent hydrolysis of MPT-AMP but only when molybdate is bound as co-substrate. MPT-AMP hydrolysis resulted in stoichiometric release of Moco that was quantitatively incorporated into plant apo-sulfite oxidase. Upon Moco formation AMP is released as second product of the reaction. When comparing MPT-AMP hydrolysis with the formation of Moco and AMP a 1.5-fold difference in reaction rates were observed. Together with the strict dependence of the reaction on molybdate the formation of adenylated molybdate as reaction intermediate in the nucleotide-assisted metal transfer reaction to molybdopterin is proposed
Llamas, A., Otte, T., Multhaup, G., Mendel, R. R., and Schwarz, G., The mechanism of nucleotide-assisted molybdenum insertion into molybdopterin - A novel route toward metal cofactor assembly, Journal of Biological Chemistry, 2006, 281, 18343-18350.
Fischer, K., Llamas, A., Tejada-Jimenez, M., Schrader, N., Kuper, J., Ataya, F. S., Galvan, A., Mendel, R. R., Fernandez, E., and Schwarz, G., Function and structure of the molybdenum cofactor carrier protein from Chlamydomonas reinhardtii, Journal of Biological Chemistry, 2006, 281, 30186-30194.
Enzyme-specific chaperones play a central role in the biogenesis of multisubunit molybdoenzymes by coordinating subunits assembly and molybdenum cofactor insertion. Molybdenum cofactor insertion is a tightly controlled process that involves specific interactions between the proteins that promote cofactor delivery, enzyme-specific chaperones, and the apoenzyme. In the assembly pathway of the multisubunit molybdoenzyme, membrane-bound nitrate reductase A from Escherichia coli, a NarJ-assisted molybdenum cofactor (Moco) insertion step, must precede membrane anchoring of the apoenzyme. The NarJ chaperone interacts at two distinct binding sites of the apoenzyme, one interferes and another is involved in molybdenum cofactor insertion. Two NarJ-binding sites within NarG are required to ensure productive formation of active nitrate reductase.
Vergnes, A., Pommier, J., Toci, R., Blasco, F., Giordano, G., and Magalon, A., NarJ chaperone binds on two distinct sites of the aponitrate reductase of Escherichia coli to coordinate molybdenum cofactor insertion and assembly, Journal of Biological Chemistry, 2006, 281, 2170-2176.
Mo storage protein
The Azotobacter vinelandii bacterium is outstanding in its capability of storing Mo in a special storage protein. The Mo storage protein is regulated by molybdenum at an extremely low concentration level (0-50 nm). It guarantees Mo-dependent nitrogen fixation even,under growth conditions of extreme Mo starvation. It is not related to any other known molybdoprotein. Each protein molecule can store at least 90 Mo atoms. Extended X-ray absorption fine-structure spectroscopy identified a metal-oxygen cluster bound to the Mo storage protein. This Mo storage protein is the only known noniron metal storage system in the biosphere containing a metal-oxygen cluster.