Identification and characterization of the cytoplasmic tungstate/molybdate-binding protein (Mop) from Eubacterium acidaminophilum

Abstract

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.

Bacterial transport of sulfate, molybdate, and related oxyanions

“…Some bacteria produce small cytoplasmic molybdate/tungstate Mop proteins (ca. 7 kDa), which are reported to be involved in oxyanion storage or homeostasis (Wagner et al 2000;Makdessi et al 2004).…”

Tungsten, the Surprisingly Positively Acting Heavy Metal Element for Prokaryotes

“…Labeling studies of E. acidaminophilum with 185 W indicate the presence of a 40‐kDa and a 5.5‐kDa protein when there are higher concentrations of tungstate or molybdate, as stated previously. Screening for proteins involved in putative W or Mo homeostasis in E. acidaminophilum led to the identification of a mop gene, using one of the three mop genes of Colostridium pasteurianum as a probe 115,116 . Such a protein or its derived domain has been termed “molbindin” to emphasize its molybdate‐binding ability 117 .…”

“…The deduced amino acid sequence exhibits the highest similarity (84–70%) to the Mop proteins from C. pasteurianum , Haemophilus influenzae, and Sporomusa ovata. The gene of E. acidaminophilum is cotranscribed with those of hypothetical proteins 115 . Rhodobacter capsulatus contains a molybdate‐activated promoter within the intergenic region upstream of its mop gene.…”

High-Affinity Vanadate Transport System in the CyanobacteriumAnabaena variabilisATCC 29413

“…Tungstoenzymes, including dehydrogenases, hydratases, and oxidoreductases that catalyze the oxidation of aldehydes, contain a tungstopterin cofactor similar to molybdopterin and are more common in hyperthermophilic bacteria and archaea with anaerobic metabolism than are molybdoenzymes (16,21,24,28,(39)(40)(41)53). Eubacterium acidaminophilum has two tungstoenzymes, viologen-dependent formate dehydrogenase and aldehyde dehydrogenase, and tungstate uptake is mediated by a specific ABC transporter encoded by the tupABC genes (31,32). Similar putative tungstate transport genes have been identified in the genomes of many other bacteria, although tungstoenzymes have not been characterized in most of these strains.…”