Journal:IUCrJ:S2052252521011696
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<b>Molecular Tour</b><br> | <b>Molecular Tour</b><br> | ||
Prodigiosin was synthesized in a bifurcated pathway by accumulating MBC and MAP which are then condensed by PigC. PigF was demonstrated to catalyze the last step of MBC pathway by transferring a methyl group to the hydroxyl group of HBC. We also demonstrated that the deletion of PigF results in the formation of an orange variant of prodigiosin the amount is much less than that of wild-type, this result indicates that PigC could recognize HBC but with much less efficiency compared with MBC. Thus, PigF is very important for the biosynthesis of prodigiosin taking into consideration of the amount of final product prodigiosin. To reveal how PigF catalyze the methyl transferring, here we determined two structures of PigF, the Apo-PigF and SAH bound PigF. Structure analysis and structural comparison with the structures of other methyltransferases indicate that PigF belongs to the typical O-methyltransferase. | Prodigiosin was synthesized in a bifurcated pathway by accumulating MBC and MAP which are then condensed by PigC. PigF was demonstrated to catalyze the last step of MBC pathway by transferring a methyl group to the hydroxyl group of HBC. We also demonstrated that the deletion of PigF results in the formation of an orange variant of prodigiosin the amount is much less than that of wild-type, this result indicates that PigC could recognize HBC but with much less efficiency compared with MBC. Thus, PigF is very important for the biosynthesis of prodigiosin taking into consideration of the amount of final product prodigiosin. To reveal how PigF catalyze the methyl transferring, here we determined two structures of PigF, the Apo-PigF and SAH bound PigF. Structure analysis and structural comparison with the structures of other methyltransferases indicate that PigF belongs to the typical O-methyltransferase. | ||
| - | In this study, we determined the crystal structures of <scene name='89/896622/Cv/3'>apo-PigF</scene> ([[7clu]]) and SAH bound PigF ([[7clf]]). The apo-PigF adopts an open conformation and the complex structure with product SAH adopts a closed conformation. The structural rearrangement happened specially at the catalytic site of the C terminal domain and not at the N terminal dimerization domain indicating the rearrangement is induced by the binding of SAH. The structural change induced by SAH results in formation of a tight binding pocket for SAH, and a putative substrate binding pocket for HBC at the same time suggesting that the two substrates (SAM and HBC) of the enzyme must be present at the same time to ensure the reaction fulfillment because only one substrate would induce the structural rearrangement. | + | In this study, we determined the crystal structures of <scene name='89/896622/Cv/3'>apo-PigF</scene> ([[7clu]]) and SAH bound PigF ([[7clf]]). The apo-PigF adopts an open conformation and the complex structure with product SAH adopts a closed conformation. <scene name='89/896622/Cv/5'>Overall structure of the apo-pigF homo-dimer</scene>. The helices, sheets and loops of monomer A are shown in royal blue, violet and green, respectively; monomer B is colored white. |
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| + | The structural rearrangement happened specially at the catalytic site of the C terminal domain and not at the N terminal dimerization domain indicating the rearrangement is induced by the binding of SAH. The structural change induced by SAH results in formation of a tight binding pocket for SAH, and a putative substrate binding pocket for HBC at the same time suggesting that the two substrates (SAM and HBC) of the enzyme must be present at the same time to ensure the reaction fulfillment because only one substrate would induce the structural rearrangement. | ||
The binding of product SAH induces dramatic conformational changes of PigF suggesting an induce-fit substrate binding mechanism of PigF. Further structure comparison suggests that this induce-fit substrate recognition mechanism may be generally existed in O-methyltransferases. Docking and mutation studies identified three key residues (His98, His247 and Asp248) crucial for enzyme activity. Essential function of His247 and Asp248 and structure analysis suggests both residues are involved in the activation of the substrate HBC of PigF. And the invariance of Asp248 in PigF further confirmed its essential role. The invariance and essential role of His98 in PigF suggests that it is involved in correctly positioning the substrate. Our study provides new insight into the catalytic mechanism of PigF, and revealed an induce-fit substrate recognition model for PigF, and broadened our understanding of O-methyltransferases. | The binding of product SAH induces dramatic conformational changes of PigF suggesting an induce-fit substrate binding mechanism of PigF. Further structure comparison suggests that this induce-fit substrate recognition mechanism may be generally existed in O-methyltransferases. Docking and mutation studies identified three key residues (His98, His247 and Asp248) crucial for enzyme activity. Essential function of His247 and Asp248 and structure analysis suggests both residues are involved in the activation of the substrate HBC of PigF. And the invariance of Asp248 in PigF further confirmed its essential role. The invariance and essential role of His98 in PigF suggests that it is involved in correctly positioning the substrate. Our study provides new insight into the catalytic mechanism of PigF, and revealed an induce-fit substrate recognition model for PigF, and broadened our understanding of O-methyltransferases. | ||
Revision as of 15:47, 24 November 2021
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