In vivo detection of molybdate-binding proteins using a competition assay with ModE in Escherichia coli.
Molybdenum is an important trace element as it forms the essential part of the active site in all molybdenum-containing enzymes. We have designed an assay for the in vivo detection of molybdate binding to proteins in Escherichia coli. The assay is based on (i). the molybdate-dependent transcriptional regulation of the moa operon by the ModE protein, and (ii). the competition for molybdate between ModE and other molybdate-binding proteins in the cytoplasm of E. coli. We were able to verify in vivo molybdate binding to three different bacterial proteins that are known to bind molybdate. This sensitive in vivo system allows the testing of different proteins for molybdate binding under in vivo conditions and will facilitate the identification of other cellular factors needed for molybdate binding. As a first example, we examined the eukaryotic protein Cnx1 that is involved in the last step of molybdenum cofactor biosynthesis in plants, and show that it is able to compete with ModE for molybdate in a molybdopterin-dependent fashion.
Kuper
Jochen
J
Department of Plant Biology, Technical University of Braunschweig, 38023, Braunschweig, Germany.
Meyer zu Berstenhorst
Sonja
S
Vödisch
Bernd
B
Mendel
Ralf R
RR
Schwarz
Günter
G
Boxer
David H
DH
D029681:Arabidopsis Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001667:Binding, Competitive; D037281:Calnexin / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008828:Microbiological Techniques; D008982:Molybdenum / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; C080351:CNX1 protein, Arabidopsis; D029968:Escherichia coli Proteins; C491064:ModE protein, E coli; C101050:ModE protein, bacteria; C404104:Mog protein, E coli; D014157:Transcription Factors; D037281:Calnexin; C044659:molybdate; D008982:Molybdenum