Identification and characterization of the cytoplasmic tungstate/molybdate-binding protein (Mop) from Eubacterium acidaminophilum.
The mop gene, encoding the molybdate-binding protein from Eubacterium acidaminophilum, was cloned using Clostridium pasteurianum mopI as a probe for heterologous hybridization. mop encodes a 69-amino-acid protein ( M(r) 7,328) with high sequence similarities to members of the molbindin protein family, which have been implicated in molybdenum storage and homeostasis. Northern blot analysis showed three mRNA transcripts (1.0, 1.6, and 2.6 kb) for mop. This result was obtained independent of the availability of tungstate in the growth medium. mop was overexpressed in Escherichia coli as a C-terminal Strep-tag fusion protein. On the basis of gel filtration, the native protein was a homohexamer of 48 kDa. The specificity of oxyanion binding was examined by protein mobility shift assay. Molybdate, tungstate, and chromate strongly changed the mobility of the protein in a native polyacrylamide gel, indicating the binding of these oxyanions to Mop. Other oxyanions, such as sulfate and phosphate, had no effect on Mop mobility. Mutational analysis revealed that the positive charge of the Arg-6, located in the conserved SARN region of Mop, was not directly involved in oxyanion binding.
Makdessi
Kathrin
K
Institut für Mikrobiologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle, Germany.
Fritsche
Kathrin
K
Pich
Andreas
A
Andreesen
Jan R
JR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002840:Chromates / Q000378:metabolism; D015342:DNA Probes; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D005051:Eubacterium / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D023061:Gene Order / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008982:Molybdenum / Q000378:metabolism*; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010710:Phosphates / Q000378:metabolism; D020161:Physical Chromosome Mapping; D011485:Protein Binding; D021122:Protein Subunits / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013431:Sulfates / Q000378:metabolism; D014158:Transcription, Genetic; D017973:Tungsten Compounds / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002840:Chromates; D015342:DNA Probes; D004269:DNA, Bacterial; D010710:Phosphates; D021122:Protein Subunits; D012333:RNA, Messenger; D013431:Sulfates; D017973:Tungsten Compounds; C050771:mopI protein, Clostridium pasteurianum; C044659:molybdate; D008982:Molybdenum; C045951:tungstate