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Literature summary for 7.6.2.8 extracted from

  • Mireku, S.A.; Sauer, M.M.; Glockshuber, R.; Locher, K.P.
    Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD (2017), Sci. Rep., 7, 14296 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 7.6.2.8

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of enzyme complex BtuCD-F in Escherichia coli strain BL21 (DE3) Gold Escherichia coli

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant complex of inhibitory nanobody Nb9 with enzyme BtuF complex, crystallization solution contains 100 mM Tris-HCl, pH 8.5, 400 mM MgCl2, and 33% w/v PEG4000, X-ray diffraction structrue determination and analysis at 2.7 A resolution Escherichia coli

Inhibitors

Inhibitors Comment Organism Structure
additional information the vitamin B12 transporter of Escherichia coli, BtuCD-F is used as a model system by generating nanobodies against the periplasmic binding protein BtuF for transporter inhibition. Six isolated nanobodies are expressed in Escherichia coli strain WK-6, they compete with B12 for binding to BtuF with inhibition constants between 0.001 and 0.000001 mM. Structure analysis of BtuF in complex with the most effective nanobody Nb9 revealing the molecular basis of its inhibitory function, the nanobody binds to the cobalamin-binding pocket of BtuF, where BtuC-binding would occur, overview. Enzyme binding to immobilized recombinant His-tagged nanobodies. Competitive inhibition of cobalamin (Cbl) binding to BtuF by the selected nanobodies. Comparison of the Nb9-BtuF complex to Cbl-bound BtuF structures Escherichia coli

Localization

Localization Comment Organism GeneOntology No. Textmining
membrane
-
 Escherichia coli 16020
-

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1] Escherichia coli
-
 ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]
-
 ?

Organism

Organism UniProt Comment Textmining
Escherichia coli P06609 AND P06611 AND P37028 genes btuC, btuD, and btuF encoding for vitamin B12 import system permease protein BtuC, vitamin B12 import ATP-binding protein BtuD, and vitamin B12-binding protein BtuF
-

Purification (Commentary)

Purification (Comment) Organism
preparation of spheroplasts of recombinnat enzyme in Escherichia coli strain BL21 (DE3) Gold membranes, recombinant enzyme and nanobody Nb9 in complex by BtuF pull-down affinity chromatography, and gel filtration Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1]
-
 Escherichia coli ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1]
-
 ?

Synonyms

Synonyms Comment Organism
BtuCD-F
-
 Escherichia coli
vitamin B12 transporter
-
 Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
23
-
 assay at Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Escherichia coli

Cofactor

Cofactor Comment Organism Structure
ATP
-
 Escherichia coli

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
additional information
-
 additional information kinetics of binding and dissociation of nanobody-BtuF complexes, stopped-flow fluorescence kinetics, overview Escherichia coli

General Information

General Information Comment Organism
physiological function bacterial ABC importers catalyze the uptake of essential nutrients including transition metals and metal-containing cofactors Escherichia coli