Engineering Giardia lamblia trimethylguanosine synthase (GlaTgs2) to transfer non-natural modifications to the RNA 5'-cap

Protein Engineering, Design & Selection : PEDS
Josephin Marie HolsteinAndrea Rentmeister

Abstract

Trimethylguanosine synthase from Giardia lamblia (GlaTgs2) naturally catalyzes methyl transfer from S-adenosyl-L-methionine (AdoMet) to the exocyclic N(2) atom of the 5'-cap--a hallmark of eukaryotic mRNAs. The wild-type enzyme shows substrate promiscuity and can also use the AdoMet-analog AdoPropen for allyl transfer. Here we report on engineering GlaTgs2 to enhance the activity on AdoPropen. A mutational analysis, involving an alanine scan of 10 residues located around the active site, was performed. Positions V34 and S38 were identified as mutational hot spots and analyzed in greater detail by testing NNK libraries. Kinetic analysis and thermostability measurements revealed V34A as the best variant of GlaTgs2, with a ∼10-fold improved specificity for AdoPropen. Double mutants did not yield additional improvements due to low catalytic efficiencies and thermal destabilization. Homologous Tgs enzymes from Homo sapiens and G. intestinalis were also investigated regarding their catalytic activity on AdoPropen. While neither the human wild-type (WT) enzyme nor any of its variants showed activity on AdoPropen, the homologue from G. intestinalis (GinTgs) was remarkably active on AdoPropen. Introducing the best substitution at the ho...Continue Reading

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Citations

Feb 18, 2016·Methods : a Companion to Methods in Enzymology·Fabian Muttach, Andrea Rentmeister
Dec 24, 2015·Angewandte Chemie·Fabian Muttach, Andrea Rentmeister
Dec 1, 2015·Methods : a Companion to Methods in Enzymology·Josephin M Holstein, Andrea Rentmeister
Nov 24, 2016·Organic & Biomolecular Chemistry·Fabian MuttachAndrea Rentmeister
Nov 22, 2016·Chemistry : a European Journal·Josephin M HolsteinAndrea Rentmeister
May 10, 2016·F1000Research·Lilith V J C MannackAndrea Rentmeister
Oct 23, 2018·Chemistry : a European Journal·Katja Hartstock, Andrea Rentmeister
Aug 9, 2019·Wiley Interdisciplinary Reviews. RNA·Nils MuthmannAndrea Rentmeister
Jul 19, 2018·Beilstein Journal of Organic Chemistry·Fabian MuttachAndrea Rentmeister
Dec 1, 2017·Chemical Communications : Chem Comm·Lea AnhäuserAndrea Rentmeister
Dec 11, 2020·Wiley Interdisciplinary Reviews. RNA·Jerry PelletierNahum Sonenberg
Jun 1, 2021·Methods : a Companion to Methods in Enzymology·Nils MuthmannAndrea Rentmeister
May 28, 2021·Chembiochem : a European Journal of Chemical Biology·Hannah DepmeierStephanie Kath-Schorr
Aug 5, 2017·ACS Central Science·Kristina Rau, Andrea Rentmeister
Jul 4, 2017·Biochemistry·Seth C Alexander, Neal K Devaraj
Oct 5, 2021·Chembiochem : a European Journal of Chemical Biology·Aileen PetersAndrea Rentmeister
Nov 6, 2021·Chemical Society Reviews·Kristie E Darrah, Alexander Deiters

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