QM/MM Studies of Dph5 – A Promiscuous Methyltransferase in the Eukaryotic Biosynthetic Pathway of Diphthamide

Hörberg, Johanna; Saenz‐Méndez, Patricia; Eriksson, Leif A.

doi:10.1021/acs.jcim.8b00217

Cited by 9 publications

(6 citation statements)

References 28 publications

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“…With excess levels of Dph5 being previously shown to bind and inhibit EF2, particularly when unmodified [ 13 , 19 , 24 , 58 ], cytotoxicity is expected to further increase when EF2 levels become limiting. This is exactly what we see in the composite mutant ( dph2 Δ eft2 Δ), and we therefore speculate that Dph5 –in addition to its catalytic activity as a promiscuous tetra-methylase [ 63 ] ( Fig 1 )–can negatively interfere with EF2 function and cell growth when the stepwise generation of diphthamide on EF2 is absent ( dph1 Δ- dph4 Δ) or incomplete ( dph7 Δ) [ 13 , 19 , 24 , 58 ]. Perhaps, such a regulatory rather than catalytic role for Dph5 involves binding to EF2 in order to exclude the elongation factor from functioning in mRNA translation unless licensed for full modification with diphthamide [ 11 , 13 ].…”

Section: Discussionsupporting

confidence: 69%

Importance of diphthamide modified EF2 for translational accuracy and competitive cell growth in yeast

et al. 2018

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In eukaryotes, the modification of an invariant histidine (His-699 in yeast) residue in translation elongation factor 2 (EF2) with diphthamide involves a conserved pathway encoded by the DPH1-DPH7 gene network. Diphthamide is the target for diphtheria toxin and related lethal ADP ribosylases, which collectively kill cells by inactivating the essential translocase function of EF2 during mRNA translation and protein biosynthesis. Although this notion emphasizes the pathological importance of diphthamide, precisely why cells including our own require EF2 to carry it, is unclear. Mining the synthetic genetic array (SGA) landscape from the budding yeast Saccharomyces cerevisiae has revealed negative interactions between EF2 (EFT1-EFT2) and diphthamide (DPH1-DPH7) gene deletions. In line with these correlations, we confirm in here that loss of diphthamide modification (dphΔ) on EF2 combined with EF2 undersupply (eft2Δ) causes synthetic growth phenotypes in the composite mutant (dphΔ eft2Δ). These reflect negative interference with cell performance under standard as well as thermal and/or chemical stress conditions, cell growth rates and doubling times, competitive fitness, cell viability in the presence of TOR inhibitors (rapamycin, caffeine) and translation indicator drugs (hygromycin, anisomycin). Together with significantly suppressed tolerance towards EF2 inhibition by cytotoxic DPH5 overexpression and increased ribosomal -1 frame-shift errors in mutants lacking modifiable pools of EF2 (dphΔ, dphΔ eft2Δ), our data indicate that diphthamide is important for the fidelity of the EF2 translocation function during mRNA translation.

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Section: Discussionsupporting

confidence: 69%

Importance of diphthamide modified EF2 for translational accuracy and competitive cell growth in yeast

et al. 2018

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“…Therefore, it is necessary to clarify the above fundamental questions of SnoK and SnoN. Herein, a series of QM/MM calculations have been performed, which has been widely and successfully applied for simulating the enzymatic reactions. − …”

Section: Introductionmentioning

confidence: 99%

Mechanism of Uncoupled Carbocyclization and Epimerization Catalyzed by Two Non-Heme Iron/α-Ketoglutarate Dependent Enzymes

Zhu

Liu

2019

J. Chem. Inf. Model.

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The non-heme iron/α-ketoglutarate dependent enzymes SnoK and SnoN from Streptomyces nogalater are involved in the biosynthesis of anthracycline nogalamycin. Although they have similar active sites, SnoK is responsible for carbocyclization whereas SnoN solely catalyzes the hydroxyl epimerization. Herein, we performed docking, molecular simulations, and a series of combined quantum mechanics and molecular mechanics (QM/MM) calculations to illuminate the mechanisms of two enzymes. The catalytic reactions of two enzymes occur on the quintet state surface. For SnoK, the whole reaction includes two separated hydrogen-abstraction steps and one radical addition, and the latter step is calculated to be rate limiting with an energy barrier of 21.7 kcal/mol. Residue D106 is confirmed to participate in the construction of the hydrogen bond network, which plays a crucial role in positioning the bulky substrate in a specific orientation. Moreover, it is found that SnoN is only responsible for the hydrogen abstraction of the intermediate, and no residue was suggested to be suitable for donating a hydrogen atom to the substrate radical, which further confirms the suggestion based on experiments that either a cellular reductant or another enzyme protein could donate a hydrogen atom to the substrate. Our docking results coincide with the previous structural study that the different roles of two enzymes are achieved by minor changes in the alignment of the substrates in front of the reactive ferryl-oxo species. This work highlights the reaction mechanisms catalyzed by SnoK and SnoN, which is helpful for engineering the enzymes for the biosynthesis of anthracycline nogalamycin.

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“…Moreover, the C3 atom of PHN, methyl group, and sulfur atom of SAM stayed in an almost linear configuration (177.48°), which is consistent with the S N 2 reaction mechanism in other methyltransferases. 19,51 In the intermediate state (Figure 5C), the methyl group covalently bonded to the C3 atom of PHN, while SAM converted to SAH. The calculated ΔE ‡ of TS1 from the reactant state was 8.6 kcal mol −1 (Figure 5D), and the calculated ΔE of the intermediate state was −32.7 kcal mol −1 .…”

Section: Qm/mm Study Of the Methyl Transfermentioning

confidence: 99%

QM/MM Study of the Catalytic Mechanism and Substrate Specificity of the Aromatic Substrate C-Methyltransferase Fur6

Zhao,

Moriwaki,

Noguchi

et al. 2024

Biochemistry

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In the field of medical chemistry and other organic chemistry, introducing a methyl group into a designed position has been difficult to achieve. However, owing to the vigorous developments in the field of enzymology, methyltransferases are considered potential tools for addressing this problem. Within the methyltransferase family, Fur6 catalyzes the methylation of C3 of 1,2,4,5,7-pentahydroxynaphthalene (PHN) using Sadenosyl-L-methionine (SAM) as the methyl donor. Here, we report the catalytic mechanism and substrate specificity of Fur6 based on computational studies. Our molecular dynamics (MD) simulation studies reveal the reactive form of PHN and its interactions with the enzyme. Our hybrid quantum mechanics/molecular mechanics (QM/MM) calculations suggest the reaction pathway of the methyl transfer step in which the energy barrier is 8.6 kcal mol −1 . Our free-energy calculations with a polarizable continuum model (PCM) indicate that the final deprotonation step of the methylated intermediate occurs after it is ejected into the water solvent from the active center pocket of Fur6. Additionally, our studies on the protonation states, the highest occupied molecular orbital (HOMOs), and the energy barriers of the methylation reaction for the analogs of PHN demonstrate the mechanism of the specificity to PHN. Our study provides valuable insights into Fur6 chemistry, contributing to a deeper understanding of molecular mechanisms and offering an opportunity to engineer the enzyme to achieve high yields of the desired product(s).

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QM/MM Studies of Dph5 – A Promiscuous Methyltransferase in the Eukaryotic Biosynthetic Pathway of Diphthamide

Cited by 9 publications

References 28 publications

Importance of diphthamide modified EF2 for translational accuracy and competitive cell growth in yeast

Importance of diphthamide modified EF2 for translational accuracy and competitive cell growth in yeast

Mechanism of Uncoupled Carbocyclization and Epimerization Catalyzed by Two Non-Heme Iron/α-Ketoglutarate Dependent Enzymes

QM/MM Study of the Catalytic Mechanism and Substrate Specificity of the Aromatic Substrate C-Methyltransferase Fur6

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