RESUMO
The KsgA methyltransferase is universally conserved and plays a key role in regulating ribosome biogenesis. KsgA has a complex reaction mechanism, transferring a total of four methyl groups onto two separate adenosine residues, A1518 and A1519, in the small subunit rRNA. This means that the active site pocket must accept both adenosine and N(6)-methyladenosine as substrates to catalyze formation of the final product N(6),N(6)-dimethyladenosine. KsgA is related to DNA adenosine methyltransferases, which transfer only a single methyl group to their target adenosine residue. We demonstrate that part of the discrimination between mono- and dimethyltransferase activity lies in a single residue in the active site, L114; this residue is part of a conserved motif, known as motif IV, which is common to a large group of S-adenosyl-L-methionine-dependent methyltransferases. Mutation of the leucine to a proline mimics the sequence found in DNA methyltransferases. The L114P mutant of KsgA shows diminished overall activity, and its ability to methylate the N(6)-methyladenosine intermediate to produce N(6),N(6)-dimethyladenosine is impaired; this is in contrast to a second active site mutation, N113A, which diminishes activity to a level comparable to L114P without affecting the methylation of N(6)-methyladenosine. We discuss the implications of this work for understanding the mechanism of KsgA's multiple catalytic steps.
Assuntos
Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/química , Metiltransferases/química , Adenosina/química , Adenosina/genética , Adenosina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico/genética , Cristalografia por Raios X , Metilação de DNA , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Humanos , Metiltransferases/deficiência , Metiltransferases/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Subunidades Ribossômicas Menores de Bactérias/enzimologia , Subunidades Ribossômicas Menores de Bactérias/genética , Especificidade por Substrato/genéticaRESUMO
Two C-terminally truncated variants of the small subunit of Mycobacterium tuberculosis isopropylmalate isomerase (Rv2987c; LeuD), LeuD_1-156 and LeuD_1-168, have been cloned, heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized. The crystals of LeuD_1-156 belonged to the hexagonal system (space group P6(1)22 or P6(5)22) with up to four subunits in the asymmetric unit, whereas the crystals of LeuD_1-168 belonged to the monoclinic system (space group P2(1)) with two subunits in the asymmetric unit. Both crystals diffracted X-rays to beyond 2.0 A resolution and were suitable for further crystallographic analysis.
