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

Abstract

Protein methylation is believed to occur extensively in creanarchaea. Recently, aKMT, a highly conserved crenarchaeal protein lysine methyltransferase, was identified and shown to exhibit broad substrate specificity in vitro. Here, we have constructed an aKMT deletion mutant of the hyperthermophilic crenarchaeon Sulfolobus islandicus. The mutant was viable but showed a moderately slower growth rate than the parental strain under nonoptimal growth conditions. Consistent with the moderate effect of the lack of aKMT on the growth of the cell, expression of a small number of genes, which encode putative functions in substrate transportation, energy metabolism, transcriptional regulation, stress response proteins, etc, was differentially regulated by more than twofold in the mutant strain, as compared with that in the parental strain. Analysis of the methylation of total cellular protein by mass spectrometry revealed that methylated proteins accounted for ϳ2/3 (1,158/1,751) and ϳ1/3 (591/ 1,757) of the identified proteins in the parental and the mutant strains, respectively, indicating that there is extensive protein methylation in S. islandicus and that aKMT is a major protein methyltransferase in this organism. No significant sequence preference was detected at the sites of methylation by aKMT. Methylated lysine residues, when visible in the structure, are all located on the surface of the proteins. The crystal structure of aKMT in complex with S-adenosyl-L-methionine (SAM) or S-adenosyl homocysteine (SAH) reveals that the protein consists of four ␣ helices and seven ␤ sheets, lacking a substrate recognition domain found in PrmA, a bacterial homolog of aKMT, in agreement with the broad substrate specificity of aKMT. Our results suggest that aKMT may serve a role in maintaining the methylation status of cellular proteins required for the efficient growth of the organism under certain non-optimal conditions. Molecular & Cellular

The extraordinary thermal stability of EstA from S. islandicus is independent of post translational modifications

“…There are no indications of residual methylation of SiEstAΔ m K from the mass spectrum [Fig. (B)], which is further supported by a proteomics study of methylation in S. islandicus where no methylated peptides from EstA were found in protein isolated from S. islandicus Δ akmt …”

“…The wildtype enzyme (SiEstA) was purified from the genetic host S. islandicus REY15A E233S while its unmethylated form (SiEstAΔ m K) was purified from S. islandicus Δ akmt , carrying a knockout of the gene SiRe_1449 coding for the lysine methyl transferase . Finally, E. coli SHuffleT7 that is engineered for improved disulphide bond formation was used for the expression of EcEstA.…”

“…Chu et al . showed that deleting the gene for aKMT4 in S. islandicus significantly reduced the level of methylated lysine, but only moderately impaired the growth of S. islandiscus . The identification of aKMT4 opens an opportunity for investigating the role of lysine methylation on the stability of proteins from creanarchaeal archaea independent of mesophilic expression systems.…”

Pathogenic Leptospires Modulate Protein Expression and Post-translational Modifications in Response to Mammalian Host Signals

“…Bound conjugates were detected using the SuperSignal WestPico substrate (Pierce) or Clarity Western ECL substrate (BioRad) and images acquired using a UVP Biospectrum–AC w/Bio Chemi camera (Cambridge, United Kingdom) or Bio-Rad ChemiDoc MP imaging system. The specificity of the PTM-specific antibodies has been previously reported (Chu et al, 2016 ; Hong et al, 2016 ).…”

Post-Translational Modifications Aid Archaeal Survival

“…This enzyme catalyzes extensive protein lysine methylation in the hyperthermophile S. islandicus, despite its being dispensable for growth. Almost all of its known substrates are nucleic acid-binding proteins, suggesting its involvement in the regulation of chromatin structure and gene expression [101].…”