The Two-Domain Structure of 5'-Adenylylsulfate (APS) Reductase from Enteromorpha intestinalis Is a Requirement for Efficient APS Reductase Activity

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• 作者：Sung-Kun Kim ; Varinnia Gomes ; Yu Gao ; Kala Chandramouli ; Michael K. Johnson ; David B. Knaff ; Thomas Leustek
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：January 16, 2007
• 年：2007
• 卷：46
• 期：2
• 页码：591 - 601
• 全文大小：739K
• 年卷期：v.46,no.2(January 16, 2007)
• ISSN：1520-4995

文摘

5'-Adenylylsulfate (APS) reductase from Enteromorpha intestinalis (EiAPR) is composed oftwo domains that function together to reduce APS to sulfite. The carboxyl-terminal domain functions asa glutaredoxin that mediates the transfer of electrons from glutathione to the APS reduction site on theamino-terminal domain. To study the basis for the interdomain interaction, a heterologous system wasconstructed in which the C domain of EiAPR was fused to the carboxyl terminus of the APS reductasefrom Pseudomonas aeruginosa (PaAPR), an enzyme that normally uses thioredoxin as an electron donorand is incapable of using glutathione for this function. The hybrid enzyme, which retains the [4Fe-4S]cluster from PaAPR, was found to use both thioredoxin and glutathione as an electron donor for APSreduction. The ability to use glutathione was enhanced by the addition of Na₂SO₄ to the reaction buffer,a property that the hybrid enzyme shares with EiAPR. When the C domain was added as a separatecomponent, it was much less efficient in conferring PaAPR with the ability to use glutathione as anelectron donor, despite the fact that the separately expressed C domain functioned in two activities thatare typical for glutaredoxins, hydroxyethyl disulfide reduction and electron donation to ribonucleotidereductase. These results suggest that the physical connection of the reductase and C domain on a singlepolypeptide is critical for the electron-transfer reaction. Moreover, the effect of Na₂SO₄ suggests that awater-ordering component of the reaction milieu is critical for the catalytic function of plant-type APSreductases by promoting the interdomain interaction.