Crystal structure and catalytic mechanism of the essential m¹ G37 tRNA methyltransferase TrmD from Pseudomonas aeruginosa
Name
RNA-2019-Jaroensuk-1481-96.pdf
Description
Published version
Size
7.58 MB
Format
Adobe PDF
Checksum (MD5)
da037719a3948ac44ad7ba3614145094
Author(s)
Prestwich, Erin
DeMott, Michael S.
Dedon, Peter C
Date Issued
March 2020
Journal
RNA (New York, N.Y.)
Publisher
Cold Spring Harbor Laboratory
Citation
Jaroensuk, Juthamas et al. “Crystal structure and catalytic mechanism of the essential m¹ G37 tRNA methyltransferase TrmD from Pseudomonas aeruginosa.” RNA (New York, N.Y.), vol. 25, no. 11, 2019, pp. 1481-1496 © 2019 The Author(s)
Version
Final published version
Abstract
© 2019 Cold Spring Harbor Laboratory Press. All rights reserved. The tRNA (m1G37) methyltransferase TrmD catalyzes m1G formation at position 37 in many tRNA isoacceptors and is essential in most bacteria, which positions it as a target for antibiotic development. In spite of its crucial role, little is known about TrmD in Pseudomonas aeruginosa (PaTrmD), an important human pathogen. Here we present detailed structural, substrate, and kinetic properties of PaTrmD. The mass spectrometric analysis confirmed the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as PaTrmD substrates. Analysis of steady-state kinetics with S-adenosyl-L-methionine (SAM) and tRNALeu(GAG) showed that PaTrmD catalyzes the two-substrate reaction by way of a ternary complex, while isothermal titration calorimetry revealed that SAM and tRNALeu(GAG) bind to PaTrmD independently, each with a dissociation constant of 14± 3 μM. Inhibition by the SAM analog sinefungin was competitive with respect to SAM (Ki = 0.41 ± 0.07 μM) and uncompetitive for tRNA (Ki =6.4 ± 0.8 μM). A set of crystal structures of the homodimeric PaTrmD protein bound to SAM and sinefungin provide the molecular basis for enzyme competitive inhibition and identify the location of the bound divalent ion. These results provide insights into PaTrmD as a potential target for the development of antibiotics.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
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Creative Commons Attribution NonCommercial License 4.0
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DOI of Published Version
https://doi.org/10.1261/RNA.066746.118
