Structural Characterization and Comparative Phylogenetic Analysis of Escherichia coli HemK, a Protein (N5)-glutamine Methyltransferase

Abstract

Protein glutamine methylation at GGQ sites of protein chain release factors plays a pivotal role in the termination of translation. We report here the crystal structure of the Escherichia coli HemK protein (N5)-glutamine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy). HemK contains two domains: a putative substrate binding domain at the N terminus consisting of a five helix bundle and a seven-stranded catalytic domain at the C terminus that harbors the binding site for AdoHcy. The two domains are linked by a β-hairpin. Structure-guided sequence analysis of the HemK family revealed 11 invariant residues functioning in methyl-donor binding and catalysis of methyl transfer. The putative substrate-binding domains of HemK from E. coli and Thermotoga maritima are structurally similar, despite the fact that they share very little sequence similarity. When the two proteins are aligned structurally, the helical N-terminal domain is subject to approximately 10° of hinge movement relative to the Cterminal domain. The apparent hinge mobility of the two domains may reflect functional importance during the reaction cycle. Comparative phylogenetic analysis of the hemK gene and its frequent neighbor gene, prfA, which encodes a major substrate, provides evidence for several examples of lateral gene transfer.

Deficiency in a Glutamine-Specific Methyltransferase for Release Factor Causes Mouse Embryonic Lethality

“…The NPPY motif found in the active site of both glutamine-and adenine-specific methyltransferases makes major contacts with the target amide group in the substrate and the methyl donor AdoMet, in common to both types of the enzymes (27). This misidentification of N6amt1 resembles the case of Dnmt2, which was initially classified as a putative cyimmunopurified with anti-eRF1 polyclonal antibodies from transfected HEK-293T cells, separated, and in-gel digested with chymotrypsin prior to MALDI-TOF MS analysis.…”

“…The murine N6amt1 gene consists of 6 exons that form two transcript isoforms with the first 4 exons in common (23). Proteins encoded by both isoforms contain the conserved GXGXG motif essential for the binding of AdoMet and the NPPY motif essential for the binding of an amide side chain (27). To ensure functional loss of the N6amt1 gene, exon 4 encoding the NPPY motif was replaced by a Neo gene through homologous recombination in embryonic stem (ES) cells (Fig.…”

The Yeast Translation Release Factors Mrf1p and Sup45p (eRF1) Are Methylated, Respectively, by the Methyltransferases Mtq1p and Mtq2p

“…1). Although overall sequence homology is relatively low, these proteins are highly homologous in the regions responsible for binding AdoMet (40), particularly motif I (44) with consensus sequence hh(D/E)hGXGXG (where h is a hydrophobic residue) and the NPPY motif, which were both previously thought to be characteristic of DNA methyltransferases (45)(46)(47). Multiple sequence alignments and phylogenic analysis demonstrated that E. coli PrmC and S. cerevisiae Mtq1p and Mtq2p, as well as their various orthologs, could be classified into two related subfamilies of protein methyltransferases (supplemental Fig.…”

Substrate Specificity of the HEMK2 Protein Glutamine Methyltransferase and Identification of Novel Substrates

“…This led to the suggestion that HemK was itself an AdoMet-dependent DNA MTase (15), and its classification in data bases as "probable MTase modifying N6-adenine or N4-cytidine in DNA" including the renaming of many entries to N6AMT. However, no evidence could be found that HemK is able to methylate DNA (16,17). Later it was found that E. coli HemK methylates a glutamine residue in the ribosomal release factors (RF1 and RF2) at the universally conserved tripeptide GGQ motif (18,19), indicating that the alignment based assignment of HemK as an AdoMet dependent MTase was correct although the predicted substrate was wrong.…”

“…The first structures of HemK MTases have been determined from Thermotoga maritima (23) and E. coli (16). Closely similar, the structures revealed two domains: an N-terminal domain with no significant similarity to MTase sequences other than close HemK homologs and a C-terminal domain that contains the (D/N/S)PP(Y/FW) motif and shows strong similarity to members of the family 7 ␤-strand methyltransferases (also referred as class I methyltransferases).…”