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mRNA Cap (Guanine-N7) Methyltransferase (Ecm1)
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Biological role
Structure
Interaction
Cap Analog Binding (Guanosine 5'-Triphosphate)
A cap analog binds to the enzyme in a pocket near AdoHcy.
6 amino acids (Tyr 145, Leu216, Leu217, Asp218, Ser219, Tyr284) are involded in the binding of the cap analog, more precisely they interact with guanine N1, N3, and O6 atoms and with the guanine exocyclic 2-NH2.
The cap makes Van der Walls contacts with side chains from Leu216, Leu217, Asp218, Ser219.
GTP makes a hydrogen bond with Tyr284 and a water mediated bond with Tyr145. [1]
As we can see on the figure above[2] , the enzyme specifically binds to guanine.
This specificity is achieved through different recognitions. The N-1 atom of adenine is unprotonated, this prevent the interaction of adenine with Ecm1. Ecm1 contact the O6 atom of guanine and permit an additional discrimination between guanine and adenine. Moreover the fact that ITP is not a substrate for Ecm1 show that the interactions between Ecm1 and guanine exocyclic 2-NH2 are important for substrate binding.
We also remark that the methyltransferase is not able to discrminate between ribose and desoxyribose nucleoside sugars.
AdoHcy Binding (S-Adenosyl-L-Homocysteine)
The mRNA Cap Methyltransferase bind to AdoHcy which is the product of the methyl donor AdoMet after the methylation.
AdoHcys is in a pocket formed by amino acids of segment 2.
10 amino acids (Lys54, Gly72, Asp78, Asp94, Ile95, Asp122, Ser124, Gln140, Phe141, Ser142) are involved in the stabilisation of AdoHcys.
The interactions between AdoHcys and the enzyme are made of :
- Hydrogen bonds mediated by Lys54, Gly72, Asp94, Asp122, Gln140
- Van der Walls interactions mediated by Ile95, Tyr124 and Ser142
- An electrostatic interaction mediated by Gln140 and Phe141
Mechanism
This enzyme catalyse N-methyl transfer from AdoMet (S-adenosylmethionine) to GpppRNA, this reaction produce 7-methyl-GpppRNA and AdoHcy. This reaction is made through a SN2 mechanism.
We remark that there is no contact between the enzyme and the guanine N-7 nucleophile, the AdoHcy sulfur leaving group or the AdoMet methyl carbon.
Indeed the enzyme does not stabilize the transition state of the chemical reaction, does not promote the activation of the nucleophile or the expulsion of the leaving group. mRNA Cap Methyltransferase brings the two substrates closer and orientates the substrates to facilitate the methyl transfer. [1]
Inhibition
- AdoHcy
It was show that the methylation of GTP increase with the concentration of AdoMet (Km of 25μM) and it was determined that the product AdoHcy has a similar affinity than AdoMet. But the activity of Ecm1 is inhibited by AdoHcy in a concentration-dependant way and the apparent IC50 was 4μm.[2]
- Sinefugin
Sinefugin, an analog of AdoMet differs from AdoMet only in the S-CH3 which is replaced by a C-NH2. It was show that sinefugin inhibit Ecm1 in a concentration-dependant manner too, the apparent IC50 is 1.5μm. The Ecm1 has an affinity for sinefugin 2/3 fold higher than for AdoMet and AdoHcy. Sinefugin has been shown to have antifungal, antiprotozoal and antiviral activities, these activities is probably related to his capacity to inhibit a variety of AdoMet-dependent methyltransferase.[2]
- Aza-AdoMet & carbocyclic aza-AdoMet
Aza-AdoMet and carbocyclic aza-AdoMet are analogs of AdoMet too. In these two molecules the sulfur atom is replaced by nitrogen. And in the carbocyclic derivate the O4-atom of the ribose is replaced by a methylene group. These two molecules are weak inhibitorsof Ecm1. The IC50 values of Aza-AdoMet is 100μm and of carbocyclic aza-AdoMet is 35μm [2]
References
- ↑ 1.0 1.1 1.2 Fabrega C, Hausmann S, Shen V, Shuman S, Lima CD. Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol Cell. 2004 Jan 16;13(1):77-89. PMID:14731396
- ↑ 2.0 2.1 2.2 2.3 Hausmann S, Zheng S, Fabrega C, Schneller SW, Lima CD, Shuman S. Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis. J Biol Chem. 2005 May 27;280(21):20404-12. Epub 2005 Mar 9. PMID:15760890 doi:10.1074/jbc.M501073200
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