Extensive Lysine Methylation in Hyperthermophilic Crenarchaea: Potential Implications for Protein Stability and Recombinant Enzymes

Abstract

In eukarya and bacteria, lysine methylation is relatively rare and is catalysed by sequence-specific lysine methyltransferases that typically have only a single-protein target. Using RNA polymerase purified from the thermophilic crenarchaeum Sulfolobus solfataricus, we identified 21 methyllysines distributed across 9 subunits of the enzyme. The modified lysines were predominantly in α-helices and showed no conserved sequence context. A limited survey of the Thermoproteus tenax proteome revealed widespread modification with 52 methyllysines in 30 different proteins. These observations suggest the presence of an unusual lysine methyltransferase with relaxed specificity in the crenarchaea. Since lysine methylation is known to enhance protein thermostability, this may be an adaptation to a thermophilic lifestyle. The implications of this modification for studies and applications of recombinant crenarchaeal enzymes are discussed.

A Prototypic Lysine Methyltransferase 4 from Archaea with Degenerate Sequence Specificity Methylates Chromatin Proteins Sul7d and Cren7 in Different Patterns

“…Cren7 methylation was completely abolished when all 12 lysine residues were changed, indicating that aKMT4 is specific to lysine. These results suggest that aKMT4 is a promiscuous protein MTase with extremely low sequence bias, which is consistent with the variegated methylation pattern of Cren7 and a number of other proteins in Sulfolobus cells (28).…”

“…We designated it as aKMT4 (belonging to the KMT4/Dot1 family of class I AdoMet-dependent MTases). Recently, Chu et al (27) also isolated and characterized the same enzyme from Sulfolobus (designated as aKMT by them) and found that it methylates Cren7 and many proteins involved in DNA replication in vitro, which correlates well with the extensive lysine methylation pattern in Sulfolobus cells (28). Our study shows that aKMT4 bears self-methylation activity and competes for a methyl group with other substrates.…”

“…However, lysine methylation is prevalent in Crenarchaea within a number of proteins bearing variegated methylation at multiple sites. For example, 21 methyl-lysines have been identified from nine subunits of the RNA polymerase complex purified from Sulfolobus solfataricus (28). aKMT4, also described by Chu et al (27), has the properties of the MTase speculated to exist by Botting et al (28).…”

“…For example, 21 methyl-lysines have been identified from nine subunits of the RNA polymerase complex purified from Sulfolobus solfataricus (28). aKMT4, also described by Chu et al (27), has the properties of the MTase speculated to exist by Botting et al (28). Most of the class I MTases are known to act on nucleic acid or chromatin proteins to regulate DNA metabolic processes, such as gene expression, RNA processing, and DNA repair (3, 4, 6).…”

“…When we compared the context sequences of lysine residues in all known substrate and nonsubstrate proteins of aKMT4, there were no apparent conserved motifs. Native Cren7 has been identified to be monomethylated at up to five sites among its 12 lysine residues in Sulfolobus cells (28). In both native Cren7 and recombinant proteins catalyzed by aKMT4, some lysine sites, including Lys-16, -24, and -31, were more frequently identified as being monomethylated through MS/MS analysis (supplemental Fig.…”