Thermal Stability and Aggregation of Sulfolobus solfataricus β-Glycosidase Are Dependent upon the N-∈-Methylation of Specific Lysyl Residues

Abstract

Methylation in vivo is a post-translational modification observed in several organisms belonging to eucarya, bacteria, and archaea. Although important implications of this modification have been demonstrated in several eucaryotes, its biological role in hyperthermophilic archaea is far from being understood. The aim of this work is to clarify some effects of methylation on the properties of ␤-glycosidase from Sulfolobus solfataricus, by a structural comparison between the native, methylated protein and its unmethylated counterpart, recombinantly expressed in Escherichia coli. Analysis by Fourier transform infrared spectroscopy indicated similar secondary structure contents for the two forms of the protein. However, the study of temperature perturbation by Fourier transform infrared spectroscopy and turbidimetry evidenced denaturation and aggregation events more pronounced in recombinant than in native ␤-glycosidase. Red Nile fluorescence analysis revealed significant differences of surface hydrophobicity between the two forms of the protein. Unlike the native enzyme, which dissociated into SDS-resistant dimers upon exposure to the detergent, the recombinant enzyme partially dissociated into monomers. By electrospray mapping, the methylation sites of the native protein were identified. A computational analysis of ␤-glycosidase three-dimensional structure and comparisons with other proteins from S. solfataricus revealed analogies in the localization of methylation sites in terms of secondary structural elements and overall topology. These observations suggest a role for the methylation of lysyl residues, located in selected domains, in the thermal stabilization of ␤-glycosidase from S. solfataricus.

Posttranslational Protein Modification inArchaea

“…In the case of Sulfolobus solfataricus ␤-glycosidase, N-ε-methylation of specific Lys residues was associated with increased thermal stability as well as with a lower susceptibility to denaturation and aggregation, in comparison to the nonmethylated recombinant version of the enzyme produced in Escherichia coli (117). The methylated Lys residues found in the Sulfolobus solfataricus enzyme are not conserved in other mesophilic glycosidases belonging to glycosyl hydrolase family I, again pointing to a thermostabilizing role for this posttranslational modification.…”

“…Methylated Lys residues have been detected in several thermophilic archaeal proteins, such as Sulfolobus acidocaldarius (485), and ␤-glycosidase (117). In the case of Sulfolobus solfataricus ␤-glycosidase, N-ε-methylation of specific Lys residues was associated with increased thermal stability as well as with a lower susceptibility to denaturation and aggregation, in comparison to the nonmethylated recombinant version of the enzyme produced in Escherichia coli (117).…”

Identification and Characterization of a Highly Conserved Crenarchaeal Protein Lysine Methyltransferase with Broad Substrate Specificity

“…Among the methylated proteins are those involved in chromosomal organization (e.g., Cren7 and Sul7d), DNA replication (e.g., Pri1/Pri2 and RFC), and transcription (RNA polymerase) (7). The roles of the modification are largely unknown, although it has been shown that methylated proteins are more stable to thermal denaturation (20,38). Since protein lysine methyltransferases such as aKMT are likely responsible for the extensive protein methylation in crenarchaea, a better understanding of these enzymes will shed considerable light on the physiological function of the posttranslational protein modification.…”

“…The early examples included ferredoxin from Sulfolobus acidocaldarius and glutamate dehydrogenase, aspartate aminotransferase, ␤-glycosidase, and ribosomal proteins from Sulfolobus solfataricus (20,38,40,45,46,55). Methylation of lysine residues in the S. solfataricus glutamate dehydrogenase and ␤-glycosidase has been suggested to enhance thermal stability of the enzymes or reduce their susceptibility to denaturation and aggregation.…”

The Ribosomal L1 Protuberance in Yeast Is Methylated on a Lysine Residue Catalyzed by a Seven-β-strand Methyltransferase

“…The chemical introduction of ⑀-dimethyllysine residues in bovine trypsin results in decreased autolysis (47) and in enhanced surface contacts in crystal structures (48). The thermal stability of ␤-glycosidase from the thermophilic archaeon Sulfolobus solfataricus is dependent upon the methylation of five of the 23 lysine residues (49). It has also been suggested that lysine side chain methylation generally increases the stability of proteins, exemplified by the extensive modification of several proteins in the thermophilic archaeon Thermoproteus tenax, where 52 methylated lysine residues were detected in 30 different proteins (50).…”