Characterization of Streptococcus pneumoniae TrmD, a tRNA methyltransferase essential for growth.
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, tRNA(Leu)(CAG). Other heterologous nonsubstrate tRNA species, like, tRNA (Thr)(GGT), tRNA(Phe), and tRNA (Ala)(TGC), bind the enzyme with similar affinities, suggesting that tRNA specificity is achieved via a postbinding event(s).
O'Dwyer
Karen
K
Microbial, Musculoskeletal and Proliferative Diseases CEDD, GlaxoSmithKline, Collegeville, Pennsylvania 19426, USA.
Watts
Joseph M
JM
Biswas
Sanjoy
S
Ambrad
Jennifer
J
Barber
Michael
M
Brulé
Hervé
H
Petit
Chantal
C
Holmes
David J
DJ
Zalacain
Magdalena
M
Holmes
Walter M
WM
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009876:Operon; D012343:RNA, Transfer / Q000138:chemical synthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000254:growth & development; D012359:tRNA Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D012343:RNA, Transfer; D012359:tRNA Methyltransferases