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Molybdate pumping into the molybdenum storage protein via an ATP-powered piercing mechanism

The molybdenum storage protein (MoSto) deposits large amounts of molybdenum as polyoxomolybdate clusters in a heterohexameric (alpha beta)(3) cage-like protein complex under ATP consumption. Here, we suggest a unique mechanism for the ATP-powered molybdate pumping process based on X-ray crystallography, cryoelectron microscopy, hydrogen-deuterium exchange mass spectrometry, and mutational studies of MoSto from Azotobacter vinelandii. First, we show that molybdate, ATP, and Mg2+ consecutively bind into the open ATP-binding groove of the beta-subunit, which thereafter becomes tightly locked by fixing the previously disordered N-terminal arm of the alpha-subunit over the beta-ATP. Next, we propose a nucleophilic attack of molybdate onto the gamma-phosphate of beta-ATP, analogous to the similar reaction of the structurally related UMP kinase. The formed instable phosphoric-molybdic anhydride becomes immediately hydrolyzed and, according to the current data, the released and accelerated molybdate is pressed through the cage wall, presumably by turning aside the Met beta 149 side chain. A structural comparison between MoSto and UMP kinase provides valuable insight into how an enzyme is converted into a molecular machine during evolution. The postulated direct conversion of chemical energy into kinetic energy via an activating molybdate kinase and an exothermic pyrophosphatase reaction to overcome a proteinous barrier represents a novelty in ATP-fueled biochemistry, because normally, ATP hydrolysis initiates large-scale conformational changes to drive a distant process.

S. Brunle, M. L. Eisinger, J. Poppe, D. J. Mills, J. D. Langer, J. Vonck, and U. Ermler,Molybdate pumping into the molybdenum storage protein via an ATP-powered piercing mechanism, Proceedings of the National Academy of Sciences of the United States of America, 2019, 116, 26497-26504.

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 MoO4. 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, 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.

Fenske, D., Gnida, M., Schneider, K., Meyer-Klaucke, W., Schemberg, J., Henschel, V., Meyer, A. K., Knochel, A., and Muller, A., A new type of metalloprotein: The mo storage protein from Azotobacter vinelandii contains a polynuclear molybdenum-oxide cluster, Chembiochem, 2005, 6, 405-413.

Molybdate binding proteins

Review including reference to molybdate binding proteins.

Souza, A. L. F., Chubatsu, L. S., Souza, E. M., Pedrosa, F. O., Monteiro, R. A., Rego, F. G. M., and Rigo, L. U., Expression, purification and DNA-binding activities of two putative ModE proteins of Herbaspirillum seropedicae (Burkholderiales, Oxalobacteraceae), Genetics and Molecular Biology, 2008, 31, 743-750.

Molybdate transporter

Fernandez-Reyes, E., Tejada-Jimenez, M., Llamas, A., Chamizo-Ampudia, A., and Galvan, A., Specific molybdate transporters and molybdoenzymes in Chlamydomonas, Febs Journal, 2012, 279, 389.

Protein binding: lanthanide salts of heteropoly molybdotungstosilicate

Lanthanide salts of heteropoly molybdotungstosilicate LnHSiMo10W2O40.xH2O (Ln = Pr, Nd, Sm, Gd, Tb, Dy, Yb) binding to bovine serum albumin: a fluorescence quenching study.

In the present work, the interaction between a series of novel lanthanide salts of heteropoly molybdotungstosilicate LnHSiMo10W2O40.xH2O (LnW2); Ln = Pr (x = 23), Nd (x = 24), Sm (x = 26), Gd (x = 20), Tb (x = 23), Dy (x = 21), Yb (x = 25), and bovine serum albumin BSA was investigated by spectroscopic approach at different temperatures under imitated physiological conditions.

In the mechanism discussion, it was proved that the fluorescence quenching of BSA by LnW2 is a result of the formation of LnW2-BSA complex.

Binding affinity between LnW2 and BSA was determined using Scatchard equation and the modified Stern-Volmer equation, and the corresponding electronic structure-affinity relationship were discussed. The results of thermodynamic parameters at different temperatures indicate that the electrostatic interactions play a major role in LnW2-BSA binding process. Moreover, the enthalpy change and entropy change were in accordance with the "enthalpy-entropy compensation" equation obtained from this and previous work.

Furthermore, the distance r between donor BSA and acceptor LnW2 was obtained according to fluorescence resonance energy transfer.

Bai, Ai Min, Ou-Yang, Yu, Yue, Hua Li, Li, Xiao Ling, and Hu, Yan Jun, Lanthanide Salts of Heteropoly Molybdotungstosilicate LnHSiMo10W2O40.xH2O (Ln = Pr, Nd, Sm, Gd, Tb, Dy, Yb) Binding to Bovine Serum Albumin: A Fluorescence Quenching Study, Biological Trace Element Research, 2012, 147, 359-365.

DNA binding
Molybdenum(VI) complex

The nuclease activity of the oxo-peroxo molybdenum(VI) complex of DNA has been studied by UV-Vis. fluorescence. viscosity, and gel electrophoresis techniques whereby the complex has been found to promote cleavage of pUC19 DNA from the supercoiled form 1 (SC) to nicked circular relaxed form II (NC). The intrinsic binding constant of the complex with DNA was calculated and found to be (15.3 ± 0.02) x 105 M-1 from UV-Vis Studies. Fluorimetric studies indicate that the complex competes with EB in binding to DNA. The relative viscosity of CT DNA increases with increasing Mo complex indicating intercalative binding. All the above results suggest that the complex binds to DNA intercalatively and imparts DNA cleavage.

Selim, M. and Mukherjea, K. K., The Nuclease Activity of an Oxo-peroxo Molybdenum Complex, Journal of Biomolecular Structure & Dynamics, 2009, 26, 561-566. Bacterial growth enhancer (BGE) molecule associated with molybdenum

Blood sera contain a previously undescribed small bacterial growth enhancer molecule (molar mass 1000-3000 Da) associated with magnesium and molybdenum ions. The addition of EDTA to the tissue culture medium lowered the growth rate, whereas the addition of bacterial growth enhancer molecule restored the growth activity.

Okayama, K., Honda, T., Matsuda, S., Saito, T., and Kawase, M., Demonstration and Partial Characterization of a Bacterial Growth Enhancer in sera, Current Microbiology, 2011, 62, 90-95.

Molybdenum and protein
Pepsin cleavage by ammonium heptamolybdate tetrahydrate

When a mixture of the porcine pepsin protein and ammonium heptamolybdate tetrahydrate (NH4)6Mo7O24·4H2O was incubated at 37 C for 24h the pepsin cleaved at Leu 112-Tyr 113, Leu 166-Leu 167 and Leu 178-Asn 179. The cleavage reaction occurs after incubation of the mixture for only 2h. The specificity of the cleavage decreases when the incubation time is longer than 48h. There was no cleavage without molybdate.

Yenjai, S., Malaikaew, P., Liwporncharoenvong, T., Buranaprapuk, A., Biochemical and biophysical research communications, 2012419126-9 Selective cleavage of pepsin by molybdenum metallopeptidase.

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