In vitro molybdenum ligation to molybdopterin using purified components.
We have previously shown that Escherichia coli MoeA and MogA are required in vivo for the final step of molybdenum cofactor biosynthesis, the addition of the molybdenum atom to the dithiolene of molybdopterin. MoeA was also shown to facilitate the addition of molybdenum in an assay using crude extracts from E. coli moeA(-) cells. The experiments detailed in this report utilized an in vitro assay for MoeA-mediated molybdenum ligation to de novo synthesized molybdopterin using only purified components and monitoring the reconstitution of human aposulfite oxidase. In this assay, maximum activation was achieved by delaying the addition of aposulfite oxidase to allow for adequate molybdenum coordination to occur. Tungsten, which substitutes for molybdenum in hyperthermophilic organisms, could also be ligated to molybdopterin using this system, though not as efficiently as molybdenum. Addition of thiol compounds to the assay inhibited activity. Addition of MogA also inhibited the reaction. However, in the presence of ATP and magnesium, addition of MogA to the assay increased the level of aposulfite oxidase reconstitution beyond that observed with MoeA alone. This effect was not observed in the absence of MoeA. The results presented here demonstrate that MoeA is responsible for mediating molybdenum ligation to molybdopterin, whereas MogA stimulates this activity in an ATP-dependent manner.
Nichols
Jason D
JD
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Rajagopalan
K V
KV
D000255:Adenosine Triphosphate / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D003067:Coenzymes; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000737:chemistry; D008667:Metalloproteins; D008982:Molybdenum / Q000737:chemistry* / Q000302:isolation & purification; D000090006:Molybdenum Cofactors; D009942:Organometallic Compounds / Q000737:chemistry* / Q000302:isolation & purification; D010088:Oxidoreductases / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011621:Pteridines / Q000737:chemistry* / Q000302:isolation & purification; D011622:Pterins / Q000737:chemistry; D013431:Sulfates / Q000737:chemistry; D013455:Sulfur / Q000737:chemistry; D013466:Sulfurtransferases / Q000378:metabolism; D013997:Time Factors; D014414:Tungsten / Q000737:chemistry
D000255:Adenosine Triphosphate; D003067:Coenzymes; D029968:Escherichia coli Proteins; D008274:Magnesium; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D009942:Organometallic Compounds; D010088:Oxidoreductases; D011621:Pteridines; D011622:Pterins; D013431:Sulfates; D013455:Sulfur; D013466:Sulfurtransferases; D014414:Tungsten; C404104:Mog protein, E coli; C022261:molybdopterin; C111302:MoeA protein, E coli