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Title: Crystal structure and catalytic mechanism of the essential m<sup>1</sup>G37 tRNA methyltransferase TrmD from Pseudomonas aeruginosa
Authors: Juthamas Jaroensuk
Yee Hwa Wong
Wenhe Zhong
Chong Wai Liew
Somchart Maenpuen
Abbas E. Sahili
Sopapan Atichartpongkul
Yok Hian Chionh
Qianhui Nah
Narumon Thongdee
Megan E. McBee
Erin G. Prestwich
Michael S. Demott
Pimchai Chaiyen
Skorn Mongkolsuk
Peter C. Dedon
Julien Lescar
Mayuree Fuangthong
Singapore-MIT Alliance for Research and Technology
NTU Institute of Structural Biology
Vidyasirimedhi Institute of Science and Technology
Chulabhorn Research Institute
Massachusetts Institute of Technology
Mahidol University
Burapha University
Tychan Ltd.
School of Biological Sciences
Chulabhorn Royal Academy
Center of Excellence on Environmental Health and Toxicology (EHT)
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 1-Jan-2019
Citation: RNA. Vol.25, No.11 (2019), 1481-1496
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.
ISSN: 14699001
Appears in Collections:Scopus 2019

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