Stabilization of tRNA (m¹G37) methyltransferase [TrmD] from Aquifex aeolicus by an intersubunit disulfide bond formation

Recombinant Aquifex aeolicus TrmD protein has a Cys20-Cys20 disulfide bond between its two subunits. This was demonstrated by SDS-polyacrylamide gel analysis of wild-type enzyme and C20S mutant protein (in which the Cys20 residue is substituted by serine), in the absence or presence of various concentrations of dithiothreitol. Analytical gel-filtration chromatography revealed that the C20S mutant protein forms a dimer structure even though it is missing the disulfide bond. Western blotting analysis suggests that the Cys20-Cys20 disulfide bond is formed in native TrmD protein in living A. aeolicus cells. Incubation at 85 °C for 20 min caused the precipitation of more than half of the C20S protein, while more than 70% of the wild-type enzyme was soluble at that temperature. This assay clearly demonstrates that the disulfide bond enhances the protein stability at 85 °C. A kinetic assay showed that the methyl-transfer activity of the C20S mutant protein was slightly less than that of the wild-type enzyme at 70 °C. Comparison of the CD-spectra of wild-type and C20S proteins reveals that some of the α-helices in the C20S mutant protein are less tightly packed than those of the wild-type enzyme at 70 °C.