Enzyme inhibition by molybdate
Novel Mode of Molybdate Inhibition of Desulfovibrio vulgaris Hildenborough
Sulfate-reducing microorganisms (SRM) are found in multiple environments and play a major role in global carbon and sulfur cycling. Because of their growth capabilities and association with metal corrosion, controlling the growth of SRM has become of increased interest. One such mechanism of control has been the use of molybdate (MoO42-), which is thought to be a specific inhibitor of SRM. The way in which molybdate inhibits the growth of SRM has been enigmatic. It has been reported that molybdate is involved in a futile energy cycle with the sulfate-activating enzyme, sulfate adenylyl transferase (Sat), which results in loss of cellular ATP. However, we show here that a deletion of this enzyme in the model SRM, Desulfovibrio vulgaris Hildenborough, remained sensitive to molybdate. We performed several subcultures of the increment sat strain in the presence of increasing concentrations of molybdate and obtained a culture with increased resistance to the inhibitor (up to 3 mM). The culture was re-sequenced and three single nucleotide polymorphisms (SNPs) were identified that were not present in the parental strain. Two of the SNPs seemed unlikely candidates for molybdate resistance due to a lack of conservation of the mutated residues in homologous genes of closely related strains. The remaining SNP was located in DVU2210, a protein containing two domains: a YcaO-like domain and a tetratricopeptide-repeat domain. The SNP resulted in a change of a serine residue to arginine in the ATP-hydrolyzing motif of the YcaO-like domain. Deletion mutants of each of the three genes apparently enriched with SNPs in the presence of inhibitory molybdate and combinations of these genes were generated in the Delta sat and wild-type strains. Strains lacking both sat and DVU2210 became more resistant to molybdate. Deletions of the other two genes in which SNPs were observed did not result in increased resistance to molybdate. YcaO-like proteins are distributed across the bacterial and archaeal domains, though the function of these proteins is largely unknown. The role of this protein in D. vulgaris is unknown. Due to the distribution of YcaO-like proteins in prokaryotes, the veracity of molybdate as a specific SRM inhibitor should be reconsidered.
G. M. Zane, J. D. Wall, and K. B. De Leon,Novel Mode of Molybdate Inhibition of Desulfovibrio vulgaris Hildenborough, Frontiers in Microbiology, 2020, 11, 610455. doi: 10.3389/fmicb.2020.610455
Purification and biochemical properties of acid phosphatase from Rohu fish liver
The aim of the study was to isolate and purify high molecular weight acid phosphatase from Rohu fish liver.
The purification processes included the enzyme precipitation by ammonium sulphate, chromatographic adsorption by CM-Cellulose, permeation chromatography on Sephadex G-100 gel and finally by affinity chromatography on Reactive Blue 4-Agarose column. The enzyme showed a purification to specific activity of 1.75 U/mg of protein with purification factor of 43 and the yield was about 0.15%.
The molecular weight was found 50 kDa on SDS-Polyacrylamide gel elecrophoresis.
The gelfiltration on calibrated Sephadex G-100 revealed molecular mass of 100 kDa indicating the dimeric nature of protein.
The K-m for p-nitrophenyl phosphate was 0.25 mM and V-max was 1.1 mu mol of substrate hydrolysed/min/mg of protein. The optimum pH for activity was 5.0. The enzyme had optimum temperature around 40 degrees C.
The enzyme exhibited broad range substrate specificity. p-NPP, phenyl phosphate, alpha-and beta-naphthyl phosphate and beta-glycero phosphate were found good substrates. Other substrates like phospho-amino acids, nucleoside phosphates and sugar phosphates were hydrolysed at reasonable rates.
The enzyme was inhibited by phosphate, fluoride, vanadate and molybdate. Competitive type of inhibition was displayed with K-i values 2.6, 0.29, 0.035 and 0.02 mM, respectively. The enzyme was also competitively inhibited by tartrate (K-i, 0.69 mM) and it saw concluded that it was recognized as tartrate sensitive acid phosphatase to distinguish it from tartrate resistant acid phosphatase class. (C) 2012 Friends Science Publishers
Siddiqua, A., Saeed, A., Naz, R., Sherazi, M., Abbas, S., and Saeed, A., Purification and Biochemical Properties of Acid Phosphatase from Rohu Fish Liver, International Journal of Agriculture and Biology, 2012, 14, 223-228.
Both arsenate and sulfate reduction were inhibited by molybdate. Arsenate-reducing bacterium, strain OREX-4, grew on lactate, with either arsenate or sulfate serving as the electron acceptor.
Newman, D.K., Kennedy, E.K., Coates, J.D., Ahmann, D., Ellis, D.J., Lovley, D.R., Morel, F.M.M. ,Dissimilatory arsenate and sulfate reduction in Desulfotomaculum auripigmentum sp. nov. Archives Of Microbiology, 1997, 168, 380-388.
Reduction of dimethylsulfoxide was not inhibited by molybdate. In Desulfovibrio desulfuricans strain PA2805, DMSO reduction occurred simultaneously-with sulfate reduction and was not effectively inhibited by molybdate, a specific inhibitor of sulfate reduction.
Jonkers, H.M., Vandermaarel, M.J.E.C., Vangemerden, H., Hansen, T.A., Dimethylsulfoxide Reduction By Marine Sulfate-Reducing Bacteria, Fems Microbiology Letters, 1996, 136, 283-287.
Townsend G.T., Ramanand K., Suflita J.M., Reductive dehalogenation and mineralization of 3-chlorobenzoate in the presence of sulfate by microorganisms from a methanogenic aquifer, Applied And Environmental Microbiology, 1997, 63, 2785-2791.
Polycyclic aromatic hydrocarbons, naphthalene and phenanthrene, were oxidized to carbon dioxide under sulfate-reducing conditions. Hydrocarbon oxidation was sulfate dependent. Molybdate, a specific inhibitor of sulfate reduction, inhibited hexadecane oxidation.
Coates, J.D., Woodward, J., Allen, J., Philp, P., Lovley, D.R., Anaerobic degradation of polycyclic aromatic hydrocarbons and alkanes in petroleum-contaminated marine harbor sediments, Applied And Environmental Microbiology,1997, 63, 3589-3593.
In anaerobic treatment of wastewater molybdate inhibited both sulfidogenesis and benzoate degradation. Molybdate inhibition increased progressively with concentration. Biogranules lost 50% of their methanogenic activity when treating waste water containing 48 mg/l of molybdate. In continuous experiments the molybdate toxicity had a threshold level. Below 0.5-0.8 mM molybdate bacterial activities were unaffected. The molybdate toxicity was not permanent. Biogranules were able gradually to regain their bioactivities once molybdate was removed from the waste water.
Liu, Y., Fang, H.H.P. Effects of molybdate on sulfate reduction and benzoate degradation, Journal Of Environmental Engineering-Asce, 1997,123 , 949-953.
Liu, S.M., Kuo, C.L. Anaerobic biotransformation of pyridine in estuarine sediments. Chemosphere, 1997, 35, 2255-2268.
Molybdate through inhibition of sulfate-reducing bacteria inhibits mercury methylation . Group VI anions, MO42-, added to sediment slurries inhibited mercury methylation: tellurate (> 50 nM )> selenate ((> 270 nM )> molybdate (100 mu M)> tungstate (700 mu M). The inhibition of mercury methylation is due to the inhibition of sulfate-reducing bacteria, which are responsible for Hg methylation. In the concentration ranges encountered in most natural aquatic environments, the inhibition of MeHg production by Group VI anions is unlikely.
Barkay, T. 1995, Methylmercury Oxidative-Degradation Potentials In Contaminated And Pristine Sediments Of The Carson River, Nevada , Applied And Environmental Microbiology, 61, 2745-2753.
Chen Y., Bonzongo J.C.J., Lyons W.B., Miller G.C., Inhibition of mercury methylation in anoxic freshwater sediment by Group VI anions, Environmental Toxicology And Chemistry, 1997, 16, 1568-1574 0730-7268.
Oremland, R.S., Miller, L.G., Dowdle, P., Connell, T., Barkay, T. 1995, Methylmercury Oxidative-Degradation Potentials In Contaminated And Pristine Sediments Of The Carson River, Nevada, Applied And Environmental Microbiology, 61, 2745-2753.
The iron oxidising bacterium T. ferrooxidans AP19-3, representative of Mo sensitive strains, could not grow on a Fe2+-medium with 1.0 mM of sodium molybdate. Mo(V), formed by reduction of Mo(VI) by Fe(II), rather than Mo(VI), is the actual inhibitor for the iron oxidation enzyme system, especially for cytochrome c oxidase. Molybdenum(V) binds to the plasma membrane and inhibits iron oxidase; as a result, growth of the bacterium is stopped [Yong et al.,1997].
Yong N.K., Oshima M., Blake R.C., Sugio T., Isolation and some properties of an iron-oxidizing bacterium Thiobacillus ferrooxidans resistant to molybdenum ion, Bioscience Biotechnology And Biochemistry, 1997, 61, 1523-1526.
Molybdate inhibited biotransformation of pyridine in sediments from the Tsengwen River.
Liu, S.M., Kuo, C.L. Anaerobic biotransformation of pyridine in estuarine sediments. Chemosphere, 1997, 35, 2255-2268.
Molybdenum enters bacteria by the active transport systems for phosphate and sulfate. Mo(V) binds erythrocytic membrane proteins (Lener and Bibr 1984)and is not mutagenic like Mo(VI). Molybdate causes irreversible cleavage of ATP to AMP by bakers yeast ATP-sulfurylase. Molybdate inhibits the activities of certain enzymes including alkaline phosphatase and NADP+ -2’ nucleotidase (Wetterhahn-Jennette 1981).
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Phosphatase inhibition by molybdate
Common bean (Phaseolus vulgaris) seedlings accumulate ureides derived from purines after germination. The first step in the conversion of purines to ureides is the removal of the 5'-phosphate group by a phosphatase that has not yes been established.
Two main phosphatase activities were detected in the embryonic axes of common bean using inosine monophosphate as substrate in an in-gel assay. Both activities differed in their sensitivity to the common phosphatase inhibitor molybdate, with the molybdate-resistant as the first enzyme induced after radicalprotrusion.
The molybdate-resistant phosphatase is the first enzyme which shows resistance to molybdate.
Cabello-Diaz, J.M., Quiles, F.A., Lambert, R., Pineda, M., Piedras, P. Plant physiology and biochemistry, 2012, 53, 54-60. Identification of a novel phosphatase with high affinity for nucleotides monophosphate from common bean (Phaseolus vulgaris).
Enzyme inhibition by molybdate
Fraqueza, G., Carvalho, L., Marques, P., Ohlin, C., Casey, W., and Aureliano, M., Functional and structural interactions of Nb, V, Mo and W oxometalates with the sarcoplasmic reticulum Ca2+ -ATPase reveal new insights into inhibition processes: a combination of NMR, Raman, AA and EPR spectroscopie with kinetic studies, Febs Journal, 2012, 279, 439.
