Journal:IUCrJ:S2052252521011696

Crystal structures of PigF, an O-methyltransferase involved in prodigiosin-synthetic pathway, reveal an induce-fit substrates recognition mechanism

Shenshen Qiu, Dongqing Xu, Mengxue Xu, Huan Zhou, Ning Sun, Li Zhang, Mengmeng Zhao, Jianhua He, Tingting Ran, Bo Sun and Weiwu Wang ^[1]

Molecular Tour
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 (7clu) and (7clf). The apo-PigF adopts an open conformation and the complex structure with product SAH adopts a closed conformation. . The helices, sheets and loops of monomer A are shown in royal blue, violet and green, respectively; monomer B is colored white. Secondary structure elements involved in the dimer interface are labelled. There is of apo-pigF. The of apo-pigF, which lacks the fragment from residues 117-152, is shown, the colors are as for chain A.

The . The dimerization domain of the subunit A is colored blue, catalytic domain is colored violet. The subunit B is colored white. SAH and ACT are shown as orange and yellow stick, respectively. is shown. Individual domains are colored blue and violet, respectively, the SAH (C atoms are in orange) is shown in the SAH-binding domain. 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 (SAH is colored orange, the residues are shown in violet ball-and-sticks, the water molecules are shown as red spheres, the hydrogen bonds are shown as dashed lines, the cartoon of PigF-SAH structure is shown as 90% transparency), 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 suggests that this induce-fit substrate recognition mechanism may be generally existed in O-methyltransferases. The cartoon of PigF-SAH is colored royal blue, the cartoon of apo-PigF is colored white (labels are in brackets), the SAH is orange ball-and-stick. . The residues are labelled in the same color, as the color of the protein.

(MBC is shown as white ball-of-sticks). 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. , His247, Glu275, and Glu304 in monomer A are colored in deep sky blue, while His247, Glu275, and Glu304 in monomer are colored in magenta.

References

1. ↑ Qiu S, Xu D, Xu M, Zhou H, Sun N, Zhang L, Zhao M, He J, Ran T, Sun B, Wang W. Crystal structures of PigF, an O-methyltransferase involved in the prodigiosin synthetic pathway, reveal an induced-fit substrate-recognition mechanism. IUCrJ. 2022 Mar 1;9(Pt 2):316-327. doi: 10.1107/S2052252521011696. eCollection, 2022 Mar 1. PMID:35371495 doi:http://dx.doi.org/10.1107/S2052252521011696