HemK, a class of protein methyl transferase with similarity to DNA methyl transferases, methylates polypeptide chain release factors, and hemK knockout induces defects in translational termination

Abstract

HemK, a universally conserved protein of unknown function, has high amino acid similarity with DNA-(adenine-N6) methyl transferases (MTases). A certain mutation in hemK gene rescues the photosensitive phenotype of a ferrochelatase-deficient (hemH) mutant in Escherichia coli. A hemK knockout strain of E. coli not only suffered severe growth defects, but also showed a global shift in gene expression to anaerobic respiration, as determined by microarray analysis, and this shift may lead to the abrogation of photosensitivity by reducing the oxidative stress. Suppressor mutations that abrogated the growth defects of the hemK knockout strain were isolated and shown to be caused by a threonine to alanine change at codon 246 of polypeptide chain release factor (RF) 2, indicating that hemK plays a role in translational termination. Consistent with such a role, the hemK knockout strain showed an enhanced rate of read-through of nonsense codons and induction of transfer-mRNA-mediated tagging of proteins within the cell. By analysis of the methylation of RF1 and RF2 in vivo and in vitro, we showed that HemK methylates RF1 and RF2 in vitro within the tryptic fragment containing the conserved GGQ motif, and that hemK is required for the methylation within the same fragment of, at least, RF1 in vivo. This is an example of a protein MTase containing the DNA MTase motif and also a protein-(glutamine-N5) MTase.

Adenine methylation in eukaryotes: Apprehending the complex evolutionary history and functional potential of an epigenetic modification

“…Within this superfamily, nucleic acid MTases belong to two distinct clades, one including all nucleic acid C5 MTases 58 and the other uniting enzymes catalyzing methylation of both N 4 C and N 6 A, those modifying the N2 position of guanines in RNA, as well as certain protein MTases methylating the amide group of glutamine in proteins such as the ribosomal protein L3 and peptide release factors (HemK family) 59, 60, 61. Members of the latter clade are characterized by a [DNSH]PP[YFW] motif at the C‐terminus of conserved strand‐4 of the Rossmann domain 18, 20, 21, 62 (Fig.…”

The methylator meets the terminator

“…With this connection to translational RFs, the knowledge of the methylation modification of these factors, and sequence similarities of HemK with methyltransferases, they were able to put all of the pieces together. In their article, they clearly show that mutants in hemK lack the modification of the glutamine residue in both RF1 and RF2 and that the HemK protein is sufficient to catalyze the methylation reaction in vitro (1). So, how can one now rationalize the original heme-defective phenotype seen?…”

“…This issue of PNAS presents an article by Nakahigashi et al (1) that shows how a gene product originally identified by its mutant phenotype as an enzyme of heme biosynthesis and then by sequence similarity as a possible DNA adenine-N-6-methyltransferase actually functions as a protein glutamine methyltransferase modulating the termination activity of release factors (RFs) in ribosomal protein synthesis! It provides a nice case study for why one can't rest until the biochemistry is done.…”

“…So, how can one now rationalize the original heme-defective phenotype seen? From microarray analysis, Nakahigashi et al (1) point out that the lack of HemK methylation has global effects on the expression of genes involved in aerobic and anaerobic metabolism that may explain the light sensitivity of the mutant, but it is still unclear exactly what caused the defects in heme metabolism observed, including the apparent lack of heme synthesis and the accumulation of intermediates in its biosynthetic pathway (2).…”

A preliminary analysis of the gene all0012 of Anabaena PCC 7120

“…For example, in E. coli, HemK/PrmC is thought to methylate the conserved glutamine residue within the translation release factors RF-1 and RF-2 (SGAGGQHVN) to terminate protein synthesis efficiently (Nakahigashi et al, 2002). In contrast to this permanent methylation, the glutamine residue within the cytoplasmic domains of transmembrane chemoreceptors is methylated by CheR and demethylated by CheB (Kehry and Dahlquist, 1982).…”