Characterization of the mRNA Capping Apparatus of Candida albicans

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Saccharomyces cerevisiae RNA triphosphatase (Cet1) and RNA guanylyltransferase (Ceg1) interact in vivo and in vitro to form a bifunctional mRNA capping enzyme complex. Here we show that the guanylyltransferase activity of Ceg1 is highly thermolabile in vitro (98% loss of activity after treatment for 10 min at 35°C) and that binding to recombinant Cet1 protein, or a synthetic peptide Cet1(232-265), protects Ceg1 from heat inactivation at physiological temperatures. Candida albicans guanylyltransferase Cgt1 is also thermolabile and is stabilized by binding to Cet1(232-265). In contrast, Schizosaccharomyces pombe and mammalian guanylyltransferases are intrinsically thermostable in vitro and they are unaffected by Cet1(232-265). We show that the requirement for the Ceg1-binding domain of Cet1 for yeast cell growth can be circumvented by overexpression in high gene dosage of a catalytically active mutant lacking the Ceg1-binding site (Cet1(269 -549)) provided that Ceg1 is also overexpressed. However, such cells are unable to grow at 37°C. In contrast, cells overexpressing Cet1(269 -549) in single copy grow at all temperatures if they express either the S. pombe or mammalian guanylyltransferase in lieu of Ceg1. Thus, the cell growth phenotype correlates with the inherent thermal stability of the guanylyltransferase. We propose that an essential function of the Cet1-Ceg1 interaction is to stabilize Ceg1 guanylyltransferase activity rather than to allosterically regulate its activity. We used protein-affinity chromatography to identify the COOHterminal segment of Ceg1 (from amino acids 245-459) as an autonomous Cet1-binding domain.

Section: Stabilization Of Ceg1 By Cet1 Versus Targeting Of Cet1 By Ceg1-supporting

confidence: 76%

Section: Effects Of Domain 2 Mutations On Ceg1 Function In Vivo-mentioning

confidence: 99%

An Essential Function of Saccharomyces cerevisiae RNA Triphosphatase Cet1 Is to Stabilize RNA Guanylyltransferase Ceg1 against Thermal Inactivation

Hausmann

Schwer

et al. 2001

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“…Inhibition of cap methylation, in particular, has been touted as an anti-infective strategy based on two lines of evidence: (i) raising the cellular levels of AdoHcy by genetic or pharmacological inhibition of AdoHcy hydrolase blocks replication of many viruses (3, 4), and (ii) the AdoMet analog sinefungin (an inhibitor of cap methylation in vitro) inhibits the growth of diverse viruses, fungi, and protozoan parasites (5-11). Indeed, it was shown recently that sinefungin displays selectivity in inhibiting yeast cap methyltransferases versus the human enzyme in vivo (12).The Saccharomyces cerevisiae cap methyltransferase Abd1 has been extensively characterized genetically, but biochemical and structural analyses of the yeast enzyme are not as far advanced (13)(14)(15)(16). Cellular cap methyltransferases from humans, Xenopus laevis, Candida albicans, Schizosaccharomyces pombe, and Trypanosoma brucei have also been characterized (17)(18)(19)(20)(21)(22).…”

mentioning

confidence: 99%

“…The Saccharomyces cerevisiae cap methyltransferase Abd1 has been extensively characterized genetically, but biochemical and structural analyses of the yeast enzyme are not as far advanced (13)(14)(15)(16). Cellular cap methyltransferases from humans, Xenopus laevis, Candida albicans, Schizosaccharomyces pombe, and Trypanosoma brucei have also been characterized (17)(18)(19)(20)(21)(22).…”

mentioning

confidence: 99%

Mutational Analysis of Encephalitozoon cuniculi mRNA Cap (Guanine-N7) Methyltransferase, Structure of the Enzyme Bound to Sinefungin, and Evidence That Cap Methyltransferase Is the Target of Sinefungin's Antifungal Activity

Zheng¹,

Hausmann²,

Liu

et al. 2006

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Cap (guanine-N7) methylation is an essential step in eukaryal mRNA synthesis and a potential target for antiviral, antifungal, and antiprotozoal drug discovery. Previous mutational and structural analyses of Encephalitozoon cuniculi Ecm1, a prototypal cellular cap methyltransferase, identified amino acids required for cap methylation in vivo, but also underscored the nonessentiality of many side chains that contact the cap and AdoMet substrates. Here we tested new mutations in residues that comprise the guaninebinding pocket, alone and in combination. The outcomes indicate that the shape of the guanine binding pocket is more crucial than particular base edge interactions, and they highlight the contributions of the aliphatic carbons of Phe-141 and Tyr-145 that engage in multiple van der Waals contacts with guanosine and S-adenosylmethionine (AdoMet), respectively. We purified 45 Ecm1 mutant proteins and assayed them for methylation of GpppA in vitro. Of the 21 mutations that resulted in unconditional lethality in vivo, 14 reduced activity in vitro to <2% of the wild-type level and 5 reduced methyltransferase activity to between 4 and 9% of wild-type Ecm1. The natural product antibiotic sinefungin is an AdoMet analog that inhibits Ecm1 with modest potency. The crystal structure of an Ecm1-sinefungin binary complex reveals sinefungin-specific polar contacts with main-chain and side-chain atoms that can explain the 3-fold higher affinity of Ecm1 for sinefungin versus AdoMet or S-adenosylhomocysteine (AdoHcy). In contrast, sinefungin is an extremely potent inhibitor of the yeast cap methyltransferase Abd1, to which sinefungin binds 900-fold more avidly than AdoHcy or AdoMet. We find that the sensitivity of Saccharomyces cerevisiae to growth inhibition by sinefungin is diminished when Abd1 is overexpressed. These results highlight cap methylation as a principal target of the antifungal activity of sinefungin.The m 7 GpppN cap structure of eukaryotic messenger RNA is formed by three enzymes. RNA triphosphatase hydrolyzes the 5Ј-triphosphate end of the pre-mRNA to a diphosphate, which is capped with GMP by RNA guanylyltransferase. RNA (guanine-N7) methyltransferase then adds a methyl group from AdoMet 2 to GpppRNA to form m 7 GpppRNA and AdoHcy. This pathway is conserved in all eukaryotic organisms and many eukaryotic viruses (1). The capping enzymes are considered attractive targets for antiviral, antifungal, and antiprotozoal drug discovery (2). Inhibition of cap methylation, in particular, has been touted as an anti-infective strategy based on two lines of evidence: (i) raising the cellular levels of AdoHcy by genetic or pharmacological inhibition of AdoHcy hydrolase blocks replication of many viruses (3, 4), and (ii) the AdoMet analog sinefungin (an inhibitor of cap methylation in vitro) inhibits the growth of diverse viruses, fungi, and protozoan parasites (5-11). Indeed, it was shown recently that sinefungin displays selectivity in inhibiting yeast cap methyltransferases versus the human enzyme in vivo (12).The S...

“…The poxvirus methyltransferase is a heterodimeric protein composed of a catalytic subunit and a stimulatory subunit; the catalytic subunit contains the sites for substrate binding and catalysis but is only weakly active on its own (5)(6)(7)(8)(9)(10)(11). In contrast, cellular cap methyltransferases are monomeric enzymes that resemble the catalytic subunit of the poxvirus methyltransferase with respect to primary structure and the effects of mutations at certain essential residues that are proposed to comprise the active site (12)(13)(14)(15)(16). The Saccharomyces cerevisiae cap methyltransferase Abd1 has been extensively characterized genetically, but biochemical studies are not as far advanced (12)(13)(14)(15).…”

mentioning

confidence: 99%

Encephalitozoon cuniculi mRNA Cap (Guanine N-7) Methyltransferase

Hausmann

Zheng

Fàbrega

et al. 2005

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The Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase Ecm1 has been characterized structurally but not biochemically. Here we show that purified Ecm1 is a monomeric protein that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to GTP. The reaction is cofactor-independent and optimal at pH 7.5. Ecm1 also methylates GpppA, GDP, and dGTP but not ATP, CTP, UTP, ITP, or m The 5Ј cap of eukaryotic messenger RNA consists of 7-methyl guanosine linked via an inverted 5Ј-5Ј triphosphate bridge to the initiating nucleoside of the transcript. The cap is formed by three enzymatic reactions: (i) the 5Ј-triphosphate end of the pre-mRNA is hydrolyzed to a diphosphate by RNA 5Ј-triphosphatase; (ii) the diphosphate RNA end is capped with GMP by