title: Characterization of Streptococcus pneumoniae TrmD, a tRNA methyltransferase essential for growth.
abstract: 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).
authors.affiliation: Microbial, Musculoskeletal and Proliferative Diseases CEDD, GlaxoSmithKline, Collegeville, Pennsylvania 19426, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: O'Dwyer
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Watts
authors.forename: Sanjoy
authors.initials: S
authors.lastname: Biswas
authors.forename: Jennifer
authors.initials: J
authors.lastname: Ambrad
authors.forename: Michael
authors.initials: M
authors.lastname: Barber
authors.forename: Hervé
authors.initials: H
authors.lastname: Brulé
authors.forename: Chantal
authors.initials: C
authors.lastname: Petit
authors.forename: David J
authors.initials: DJ
authors.lastname: Holmes
authors.forename: Magdalena
authors.initials: M
authors.lastname: Zalacain
authors.forename: Walter M
authors.initials: WM
authors.lastname: Holmes
mesh_terms: 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*
chemical_list: D011994:Recombinant Proteins; D012343:RNA, Transfer; D012359:tRNA Methyltransferases