Magnesium-Induced Nucleophile Activation in the Guanylyltransferase mRNA Capping Enzyme

Swift, Robert V.; Ong, Chau D.; Amaro, Rommie E.

doi:10.1021/bi301224b

Cited by 8 publications

(12 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the hepatitis delta virus (HDV) ribozyme, which has a similar divalent metal ion requirement under physiological conditions, a pK a shift of similar magnitude (~−4 units) on the 2′-hydroxyl nucleophile induced by Mg 2+ was predicted by 3D-RISM calculations(95). Further, Amaro and co-workers have reported the Mg 2+ -induced pK a shift of lysine K82 in guanylyltransferase mRNA capping enzyme to be −4.2(94) which is very close to the result reported here using a similar TI approach. Rosta and co-workers have also observed that Mg 2+ plays the role of altering pK a in dUTPase which catalyzes phosphate hydrolysis(96, 97).…”

Section: Resultssupporting

confidence: 86%

“…The convergence of the TI simulations in terms of the target observable pK a shifts was measured (Fig. 6) and showed that the simulations reached reasonable convergence after 1 ns, which was similar to the timescales investigated in related work(56, 93, 94). Experimental activity-pH profiles indicate that the apparent pK a of the general base in HHR is around 8 in the presence of Mg 2+ (15).…”

Section: Resultssupporting

confidence: 80%

See 1 more Smart Citation

Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations

et al. 2017

View full text Add to dashboard Cite

The hammerhead ribozyme is a well-studied nucleolytic ribozyme that catalyzes the self-cleavage of the RNA phosphodiester backbone. Despite experimental and theoretical efforts, there remain key questions about details of the mechanism with regard to the activation of the nucleophile by the putative general base guanine (G12). Straight-forward interpretation of the measured activity-pH data implies the pKa value of the G12 nucleobase is significantly shifted by the ribozyme environment. Recent crystallographic and biochemical work has identified pH-dependent divalent metal ion binding at the N7/O6 position of G12, leading to the hypothesis that this binding mode could induce a pKa shift of G12 towards neutrality. We present computational results that unify the interpretation of available structural and biochemical data, and paint a detailed mechanistic picture of the general base step of the reaction. Electronic structure calculations quantify the magnitude of predicted pKa shifts induced by Mg2+ binding in several Mg2+-guanine complexes. Molecular dynamics and free energy simulations using newly developed 12-6-4 parameters for divalent metal ion binding to nucleic acids are used to characterize the ribozyme active site environment and evaluate the pKa of G12 in HHR with and without the Mg2+ ion bound. The results suggest that Mg2+ is able to down-shift the pKa of G12 by −1.2 units in accord with the apparent pKa value determined from activity-pH measurements. In addition, ab initio quantum mechanical/molecular mechanical simulations are performed to explore the free energy profile for the general base step in the presence and absence of Mg2+. Taken together, these results are in quantitative agreement with available experimental data, and support a cooperative mechanism whereby Mg2+ binding at the N7/O6 position serves to stabilize G12 in the functional, deprotonated form that can abstract a proton from the nucleophile in the general base step of the reaction. In this scenario, site-specific Mg2+ ion binding acts as a switch to activate the general base in HHR. Finally, experimentally-testable predictions are made on the mutational and rescue effects on G12, which will give further insights into the catalytic mechanism. These results contribute to our growing knowledge of the potential roles of divalent metal ions in RNA catalysis.

show abstract

Section: Resultssupporting

confidence: 86%

Section: Resultssupporting

confidence: 80%

Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations

et al. 2017

View full text Add to dashboard Cite

show abstract

“…These findings suggest that insufficient Mg in the male mgt5 and mgt9 gametophytes led to developmental defects. Magnesium is a mineral element that is important for many functions of living cells including DNA replication and transcription and enzyme activity (Jensen et al, 2000;Batra et al, 2006;Swift et al, 2012). Hence, insufficient Mg in the male gametophyte may affect DNA replication, gene expression and enzyme activity during mitosis and intine formation.…”

Section: Discussionmentioning

confidence: 99%

“…Magnesium (Mg) is a divalent metal cation that is important for numerous biological processes and cellular functions in living cells, including DNA replication and posttranscriptional processing (Batra et al, 2006;Swift et al, 2012). Magnesium uptake and its dynamic balance in the cell rely on Mg transporters.…”

Section: Introductionmentioning

confidence: 99%

Magnesium Transporter 5 plays an important role in Mg transport for male gametophyte development in Arabidopsis

Wang

Lou

et al. 2015

The Plant Journal

View full text Add to dashboard Cite

SUMMARYDuring anther development the male gametophyte develops inside the locule and the tapetal cells provide all nutrients for its development. Magnesium Transporter 5 (MGT5) is a member of the MGT family and has dual functions of Mg export and import. Here, we show that male gametophyte mitosis and intine formation are defective in a mgt5 mutant. The transient expression of GFP-MGT5 revealed that MGT5 is localized in the plasma membrane. These findings suggest that in the male gametophyte MGT5 plays a role in importing Mg from the locule and that Mg is essential for male gametophyte development. The expression of MGT5 in the knockout ABORTED MICROSPORES (AMS) mutant (AMS being an essential regulator of tapetum) is tremendously reduced. Chromatin immunoprecipitation and mobility shift assay experiments demonstrated that AMS can directly bind the promoter of MGT5. An immunoelectron microscopy assay revealed that MGT5-His is localized to the plasma membrane of the tapetum. These findings suggest that AMS directly regulates MGT5 in the tapetum and thus induces export of Mg into the locule. The mgt5 plant exhibits severe male sterility while the expression of MGT5 under the tapetum-specific promoter A9 partly rescued mgt5 fertility. mgt5 fertility was restored under high-Mg conditions. These findings suggest that the mgt5 tapetum still has the ability to export Mg and that a sufficient supply of Mg from the tapetum can improve the importation of Mg in the mgt5 male gametophyte. Therefore, MGT5 plays an important role in Mg transport from the tapetum to the microspore.

show abstract

“…18 The reaction is held in the presence of the divalent metal cation Mg 2+ , which minimizes the charge repulsion between phosphate groups, favoring nucleophile activation. 19,20 Moreover, two positively charged residues polarize these groups, increasing the nucleophilic nature of the oxygen attacking the phosphor atom. 21,22 AGPase follows a sequential ordered bi-bi mechanism with ATP binding first, followed by G1P and by the ordered release of PPi and ADPglucose.…”

Section: Introductionmentioning

confidence: 99%

The allosteric control mechanism of bacterial glycogen biosynthesis disclosed by cryoEM

Cifuente

Comino

D’Angelo

et al. 2020

Preprint

View full text Add to dashboard Cite

ABSTRACTGlycogen and starch are the major carbon and energy reserve polysaccharides in nature, providing living organisms with a survival advantage. The evolution of the enzymatic machinery responsible for the biosynthesis and degradation of such polysaccharides, led the development of mechanisms to control the assembly and disassembly rate, to store and recover glucose according to cell energy demands. The tetrameric enzyme ADP-glucose pyrophosphorylase (AGPase) catalyzes and regulates the initial step in the biosynthesis of both α-polyglucans. Importantly, AGPase displays cooperativity and allosteric regulation by sensing metabolites from the cell energy flux. The understanding of the allosteric signal transduction mechanisms in AGPase arises as a long-standing challenge. In this work, we disclose the cryoEM structures of the paradigmatic homotetrameric AGPase from Escherichia coli (EcAGPase), in complex with either positive or negative physiological allosteric regulators, FBP and AMP respectively, both at 3.0 Å resolution. Strikingly, the structures reveal that FBP binds deeply into the allosteric cleft and overlaps the AMP site. As a consequence, FBP promotes a concerted conformational switch of a regulatory loop, RL2, from a ‘locked’ to a ‘free’ state, modulating ATP binding and activating the enzyme. This notion is strongly supported by our complementary biophysical and bioinformatics evidence, and a careful analysis of vast enzyme kinetics data on single-point mutants of EcAGPase. The cryoEM structures uncover the residue interaction networks (RIN) between the allosteric and the catalytic components of the enzyme, providing unique details on how the signaling information is transmitted across the tetramer, from which cooperativity emerges. Altogether, the conformational states visualized by cryoEM reveal the regulatory mechanism of EcAGPase, laying the foundations to understand the allosteric control of bacterial glycogen biosynthesis at the molecular level of detail.

show abstract

Magnesium-Induced Nucleophile Activation in the Guanylyltransferase mRNA Capping Enzyme

Cited by 8 publications

References 50 publications

Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations

Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations

Magnesium Transporter 5 plays an important role in Mg transport for male gametophyte development in Arabidopsis

The allosteric control mechanism of bacterial glycogen biosynthesis disclosed by cryoEM

Contact Info

Product

Resources

About