The post-translational modification of EF-P K34 was initially recognized by a mass shift of +144 Da (Aoki et al., 2008). Genetic studies suggested a post-translation modification of K34 by (R)‐β‐lysine (+128 Da) required two enzymes, EpmA and EpmB (Figure 7A) (Bailly and de Crecy-Lagard, 2010). First, the lysine 2,3-aminomutase (EpmB, also referred to as YjeK) transforms (S)-α-lysine into (R)-β-lysine (Behshad et al., 2006; Roy et al., 2011).
Second, the Elongation factor P-(R)-β-lysine ligase (EpmA, also referred to as GenX, PoxA and YjeA) ligates (R)-β-lysine to EF-P K34 in an ATP-dependent manner. This mechanism was confirmed by genetic (Ambrogelly et al., 2010; Navarre et al., 2010) and structural work (Sumida et al., 2010), and by biochemical in vitro assays (Yanagisawa et al., 2010).
EpmA evolved from a class II amino acyl tRNA synthetase (aaRS) which lost its anticodon binding domain, gained an EF-P specificity and has a broad substrate spectrum (Bailly and de Crecy-Lagard, 2010; Katz et al., 2014; Navarre et al., 2010; Yanagisawa et al., 2010). In addition to the target substrate, (R)-β-lysine, in vivo studies showed that EpmA can equally well utilize the enantiomer (S)-β-lysine, which in fact leads to an activated EF-P (Gilreath et al., 2011; Roy et al., 2011). In addition, in an in-vitro assay also the constitutional isomer (L)-α-lysine was accepted and ligated to K34 of EF-P. However, the α-lysinylated EF-P is inactive (Gilreath et al., 2011; Roy et al., 2011). Notably, in a substrate competition assay (R)‐β‐lysine (KM = 213 µM) is highly favored over its α‐lysine constitutional isomer (KM = 8600 µM) and its enantiomer (KM = 6950 µM) (Roy et al., 2011). The discrepancy of +16 Da between the mass shift +144 Da and +128 Da by (R)-β-lysine corresponds to the addition of a single oxygen atom, likely, in the form of a hydroxyl moiety. Elogation factor P hydroxylase (EpmC, also referred to as YfcM) was
Introduction
reported to hydroxylate K34 of EF-P. YfcM binds only lysinylated EF-P and modifies it at the δ-C5 of K34 (Kobayashi et al., 2014a; Kobayashi et al., 2014b; Peil et al., 2012).
Because EF-P is universally conserved and its modification is of key functional importance (Doerfel et al., 2013; Navarre et al., 2010; Ude et al., 2013; Yanagisawa et al., 2010), it is surprising that only 22% of all sequenced bacterial genomes encode orthologs of EpmA or EpmB (Bailly and de Crecy-Lagard, 2010; Lassak et al., 2015). A phylogentic analysis revealed that in 9% of all sequenced bacterial genomes, a conserved arginine residue (R32) is found in a position equivalent to K34 (Choi and Choe, 2011; Lassak et al., 2015). In Shewanella oneidenis, an EF-P R32 modifying enzyme was identified. The EF-P R32 rhamnosyl-transferase (EarP) uses dTDP-β-(L)-rhamnose as a substrate; the modification of EF-P R32 with rhamnosylation was confirmed by mass-spectrometry (Figure 7B) (Lassak et al., 2015; Wang et al., 2017). Anti-R32-rhamanosyl antibodies and crystallographic studies confirmed the EF-P-EarP interactions in multiple bacterial strains (Krafczyk et al., 2017; Li et al., 2016; Sengoku et al., 2018). In contrast to the EF-P lysinylation mechanism, the usage of dTDP-(L)-rhamnose competes with many other intracellular biosynthetic pathways (Babaoglu et al., 2003; Lam et al., 2011; Lindhout et al., 2009; Rahim et al., 2001; Schirm et al., 2004). Therefore, the flow and the availability of substrate has to be under tight regulation to ensure the efficient modification of EF-P R32.
Figure 7: Post-translational modification of EF-P.
A) E. coli EF-P residue K34 modified to ε(R)-β-lysylhydroxylysine by action of EpmA and C.
B) S. oneidenis EF-P rhamnosylated at residue R32 by EarP.
The described bacterial EF-P post-translational modification machineries can only be found in 31% of all sequenced bacterial genomes and mainly in genomes of Gram-negative bacteria. Recently the post-translational modification of EF-P in the Gram-positive model organism Bacillus subtilis was identified. The modification of K32 with 5-aminopentanol showed a positive impact on the translation of poly(Pro) containing proteins. In B. subtilis EF-P-dependent peptide sequences were mainly found in flagellar genes; deletion of EF-P caused a reduced swarming motility. Initially reported sporulation failures (Meeske et al., 2016; Ohashi et al., 2003), could not be restored by complementation studies. In contrast to the previously described modification, the genes
Introduction
encoding the modification enzymes were not found in the direct neighborhood to the EF-P gene (Rajkovic et al., 2016), which makes it much harder to identify putative modifying enzyme from a genetic screening. The B. subtilis EF-P modifying enzymes Ymfl was shown to catalyze the reduction of 5-aminopentanone to 5-aminopentanol.
Interestingly, a 5-aminopenatnone modified EF-P appear to be biochemically inactive, whereas the unmodified EF-P remained active (Hummels et al., 2017). A genetic screen revealed two other genes (ynbB and gsaB) involved in the modification, however, their direct contribution to a distinct modification step could not be assigned yet. In the corresponding deletion strains K32 was acetylated instead of modified to 5-aminopentanol. Furthermore, three genes (yaaO, yfkA and ywlG) were identified which influence the degree of EF-P K32 modification, however, their mode of action remains to be elucidated (Witzky et al., 2018). The following modification pathway was suggest: K32 becomes modified with hydroxypentanone, which in turns becomes dehydrate and forms pentanone, which is converted to 5-aminopentanone by a hydroamination reaction, and reduced to 5-aminopentanol by Ymfl (Witzky et al., 2018). B. subtilis was reported to tune the activity of EF-P, although the mechanism is not yet fully understood. The deletion of EF-P in B. subtilis causes a reduced swarming motility, which is in line with the occurrence of poly(Pro) sequences in genes related to cell motility. The lack of a severe phenotype correlates with the general low abundance of poly(Pro) sequences in the B. subtilis genome. It was proposed that for bacteria with a high abundance of poly(Pro) sequences in their genome, EF-P is more important than in those with a low abundance of poly(Pro) sequences. Overall, the nature of many post-translational modifications of EF-P is yet not described. It still remains unknown whether other factors can perform similar task or compensate for the lack of EF-P. Organism with either no or two copies of EF-P have been reported, but the functional significance of these findings remains unclear (reviewed in:
(Lassak et al., 2016)). For some gammaproteobacteria, such as E. coli or Vibrio cholera, an EFP-like protein (YeiP) was described as a paralog of EF-P (Richards et al., 2012). Whether it binds to the ribosome or can compensate for the lack of EF-P remains to be elucidated.