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Journal of Bacteriology, April 2004, p. 2346-2354, Vol. 186, No. 8
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.8.2346-2354.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of Streptococcus pneumoniae TrmD, a tRNA Methyltransferase Essential for Growth

Karen O'Dwyer,¹ Joseph M. Watts,² Sanjoy Biswas,¹ Jennifer Ambrad,^1, Michael Barber,¹ Hervé Brulé,² Chantal Petit,¹ David J. Holmes,¹ Magdalena Zalacain,^1* and Walter M. Holmes^2*

Microbial, Musculoskeletal and Proliferative Diseases CEDD, GlaxoSmithKline, Collegeville, Pennsylvania 19426,¹ Institute for Structural Biology and Drug Discovery and Department of Microbiology and Immunology, Medical College of Virginia, Campus of Virginia Commonwealth University, Richmond, Virginia 23298²

Received 24 June 2003/ Accepted 5 January 2004

Down-regulation of expression of trmD, encoding the enzyme tRNA (guanosine-1)-methyltransferase, has shown that this gene is essential for growth of Streptococcus pneumoniae. The S. pneumoniae trmD gene has been isolated and expressed in Escherichia coli by using a His-tagged T7 expression vector. Recombinant protein has been purified, and its catalytic and physical properties have been characterized. The native enzyme displays a molecular mass of approximately 65,000 Da, suggesting that streptococcal TrmD is a dimer of two identical subunits. In fact, this characteristic can be extended to several other TrmD orthologs, including E. coli TrmD. Kinetic studies show that the streptococcal enzyme utilizes a sequential mechanism. Binding of tRNA by gel mobility shift assays gives a dissociation constant of 22 nM for one of its substrates, . Other heterologous nonsubstrate tRNA species, like , tRNA^Phe, and , bind the enzyme with similar affinities, suggesting that tRNA specificity is achieved via a postbinding event(s).

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* Corresponding author. Mailing address for Magdalena Zalacain: Microbial, Musculoskeletal and Proliferative Diseases CEDD, GlaxoSmithKline, 1250 South Collegeville Rd., Collegeville, PA 19426. Phone: (610) 917-6450. Fax: (610) 917-7901. E-mail: magdalena_zalacain-1{at}gsk.com. Mailing address for Walter M. Holmes: Institute for Structural Biology and Drug Discovery and Department of Microbiology and Immunology, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, VA 23298. Phone: (804) 828-2327. Fax: (804) 827-3664. E-mail: holmes{at}hsc.vcu.edu.

Present address: Department of Fisheries, Animal, and Veterinary Sciences, University of Rhode Island, Kingston, RI 02881.

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Journal of Bacteriology, April 2004, p. 2346-2354, Vol. 186, No. 8
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.8.2346-2354.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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