Stabilization of tRNA (mG37) 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.