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

Abstract

Protein lysine methylation occurs extensively in the Crenarchaeota, a major kingdom in the Archaea. However, the enzymes responsible for this type of posttranslational modification have not been found. Here we report the identification and characterization of the first crenarchaeal protein lysine methyltransferase, designated aKMT, from the hyperthermophilic crenarchaeon Sulfolobus islandicus. The enzyme was capable of transferring methyl groups to selected lysine residues in a substrate protein using S-adenosyl-L-methionine (SAM) as the methyl donor. aKMT, a non-SET domain protein, is highly conserved among crenarchaea, and distantly related homologs also exist in Bacteria and Eukarya. aKMT was active over a wide range of temperatures, from ϳ25 to 90°C, with an optimal temperature at ϳ60 to 70°C. Amino acid residues Y9 and T12 at the N terminus appear to be the key residues in the putative active site of aKMT, as indicated by sequence conservation and site-directed mutagenesis. Although aKMT was identified based on its methylating activity on Cren7, the crenarchaeal chromatin protein, it exhibited broad substrate specificity and was capable of methylating a number of recombinant Sulfolobus proteins overproduced in Escherichia coli. The finding of aKMT will help elucidate mechanisms underlining extensive protein lysine methylation and the functional significance of posttranslational protein methylation in crenarchaea.

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

“…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. S2) (27). The correlation between in vivo and in vitro methylation data suggests that aKMT4 may serve as the main MTase for Cren7 methylation in Sulfolobus.…”

“…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.…”

“…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).…”

aKMT Catalyzes Extensive Protein Lysine Methylation in the Hyperthermophilic Archaeon Sulfolobus islandicus but is Dispensable for the Growth of the Organism

“…However, no SET domain proteins have been found in the sequenced genomes of crenarchaea where lysine methylation is prevalent. Recently, we identified the first crenarchaeal protein lysine methyltransferase, designated as aKMT, from S. islandicus (35). This protein resembles methyltransferases of the eukaryotic Dot1 family (36).…”

“…This protein resembles methyltransferases of the eukaryotic Dot1 family (36). Notably, aKMT is capable of methylating several tested recombinant Sulfolobus proteins overproduced in Escherichia coli, exhibiting broad substrate specificity in vitro (35).…”