title: Engineering Giardia lamblia trimethylguanosine synthase (GlaTgs2) to transfer non-natural modifications to the RNA 5'-cap.
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 homologous position led to variant T34A with ∼40-fold higher specificity for AdoPropen than the original GlaTgs2 WT.
authors.affiliation: Westfälische Wilhelms-Universität Münster, Institute of Biochemistry, Muenster 48149, Germany.
authors.forename: Josephin Marie
authors.initials: JM
authors.lastname: Holstein
authors.affiliation: Westfälische Wilhelms-Universität Münster, Institute of Biochemistry, Muenster 48149, Germany Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), University of Muenster, Muenster 48149, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Stummer
authors.affiliation: Westfälische Wilhelms-Universität Münster, Institute of Biochemistry, Muenster 48149, Germany Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), University of Muenster, Muenster 48149, Germany a.rentmeister@uni-muenster.de.
authors.forename: Andrea
authors.initials: A
authors.lastname: Rentmeister
mesh_terms: D000409:Alanine; D004795:Enzyme Stability; D016829:Giardia lamblia / Q000201:enzymology*; D006358:Hot Temperature; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D008780:Methyltransferases; C456271:trimethylguanosine synthase; D000409:Alanine
keywords: 5'-cap; AdoMet analog; Giardia intestinalis; Giardia lamblia; trimethylguanosine synthase