Molybdate binding by ModA, the periplasmic component of the Escherichia coli mod molybdate transport system

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• 作者：Imperial ; Juan ; Hadi ; Margono ; Amy ; Nancy K.
• 关键词：ModA ; Molybdate binding ; Tungstate binding ; Molybdenum transport ; Escherichia coli
• 刊名：Biochimica et Biophysica Acta (BBA)/Biomembranes
• 出版年：1998
• 出版时间：March 13, 1998
• 年：1998
• 卷：1370
• 期：2
• 页码：337-346
• 全文大小：915 K

文摘

ModA, the periplasmic-binding protein of the Escherichia coli mod transport system was overexpressed and purified. Binding of molybdate and tungstate to ModA was found to modify the UV absorption and fluorescence emission spectra of the protein. Titration of these changes showed that ModA binds molybdate and tungstate in a 1:1 molar ratio. ModA showed an intrinsic fluorescence emission spectrum attributable to its three tryptophanyl residues. Molybdate binding caused a conformational change in the protein characterized by: (i) a shift of tryptophanyl groups to a more hydrophobic environment; (ii) a quenching (at pH 5.0) or enhancement (at pH 7.8) of fluorescence; and (iii) a higher availability of tryptophanyl groups to the polar quencher acrylamide. The tight binding of molybdate did not allow an accurate estimation of the binding constants by these indirect methods. An isotopic binding method with ⁹⁹MoO₄²⁻ was used for accurate determination of K_D (20 nM) and stoichiometry (1:1 molar ratio). ModA bound tungstate with approximately the same affinity, but did not bind sulfate or phosphate. These K_Ds are 150- to 250-fold lower than those previously reported, and compatible with the high molybdate transport affinity of the mod system. The affinity of ModA for molybdate was also determined in vivo and found to be similar to that determined in vitro. Publisher: Elsevier Science Language of Publication: English Item Identifier: S0005-2736(98)00003-0 Publication Type: Article ISSN: 0005-2736 Cited by:

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2. Delarbre, Laure; Stevenson, Clare E.M.; White, Daniel J.; Mitchenall, Lesley A.; Pau, Richard N.; Lawson, David M.,""Two crystal structures of the cytoplasmic molybdate-binding protein ModG suggest a novel cooperative binding mechanism and provide insights into ligand-binding specificity²""Journal of Molecular Biology2001pp. 1063-1079
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3. Gates, Andrew J.; Hughes, Richard O.; Sharp, Sarah R.; Millington, Paul D.; Nilavongse, Arjaree; Cole, Jeffrey A.; Leach, Emily-Rose; Jepson, Brian; Richardson, David J.; Butler, Clive S.,""Properties of the periplasmic nitrate reductases from Paracoccus pantotrophus and Escherichia coli after growth in tungsten-supplemented media""FEMS Microbiology Letters2003pp. 261-269
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5. Stewart, Lisa J.; Bailey, Susan; Bennett, Brian; Charnock, John M.; Garner, C. David; McAlpine, Alan S.,""Dimethylsulfoxide reductase: an enzyme capable of catalysis with either molybdenum or tungsten at the active site³""Journal of Molecular Biology2000pp. 593-600
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Footnotes:

1. Florida Agricultural Experiment Station Journal Series No. R-08044.