Crystal structure and catalytic mechanism of the essential m1G37 tRNA methyltransferase TrmD from Pseudomonas aeruginosa
- Juthamas Jaroensuk1,2,11,13,
- Yee Hwa Wong3,4,13,
- Wenhe Zhong2,4,
- Chong Wai Liew4,
- Somchart Maenpuen5,
- Abbas E. Sahili3,4,
- Sopapan Atichartpongkul6,
- Yok Hian Chionh2,12,
- Qianhui Nah2,
- Narumon Thongdee1,
- Megan E. McBee2,
- Erin G. Prestwich7,
- Michael S. DeMott7,
- Pimchai Chaiyen8,
- Skorn Mongkolsuk6,9,10,
- Peter C. Dedon2,7,
- Julien Lescar3,4 and
- Mayuree Fuangthong1,6,10
- 1Applied Biological Sciences Program, Chulabhorn Graduate Institute, Chulabhorn Royal Academy, Bangkok 10210, Thailand
- 2Singapore-MIT Alliance for Research and Technology Antimicrobial Resistance and Infectious Disease Interdisciplinary Research Groups, 138602 Singapore
- 3School of Biological Sciences, Nanyang Technological University, 637551 Singapore
- 4NTU Institute of Structural Biology, Nanyang Technological University, 636921 Singapore
- 5Department of Biochemistry, Faculty of Science, Burapha University, Chonburi 20131, Thailand
- 6Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok 10210, Thailand
- 7Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
- 8School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong 21210, Thailand
- 9Department of Biotechnology, Faculty of Sciences, Mahidol University, Bangkok 10400, Thailand
- 10Center of Excellence on Environmental Health and Toxicology (EHT), Bangkok 10400, Thailand
- Corresponding authors: mayuree{at}cri.or.th, julien{at}ntu.edu.sg, pcdedon{at}mit.edu
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↵13 These authors contributed equally to this work.
Abstract
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.
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Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.066746.118.
- Received June 9, 2018.
- Accepted July 28, 2019.
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