Deciphering the Translation Initiation Factor 5A Modification Pathway in Halophilic Archaea

Abstract

Translation initiation factor 5A (IF5A) is essential and highly conserved in Eukarya (eIF5A) and Archaea (aIF5A). The activity of IF5A requires hypusine, a posttranslational modification synthesized in Eukarya from the polyamine precursor spermidine. Intracellular polyamine analyses revealed that agmatine and cadaverine were the main polyamines produced in Haloferax volcanii in minimal medium, raising the question of how hypusine is synthesized in this halophilic Archaea. Metabolic reconstruction led to a tentative picture of polyamine metabolism and aIF5A modification in Hfx. volcanii that was experimentally tested. Analysis of aIF5A from Hfx. volcanii by LC-MS/MS revealed it was exclusively deoxyhypusinylated. Genetic studies confirmed the role of the predicted arginine decarboxylase gene (HVO_1958) in agmatine synthesis. The agmatinase-like gene (HVO_2299) was found to be essential, consistent with a role in aIF5A modification predicted by physical clustering evidence. Recombinant deoxyhypusine synthase (DHS) from S. cerevisiae was shown to transfer 4-aminobutyl moiety from spermidine to aIF5A from Hfx. volcanii in vitro. However, at least under conditions tested, this transfer was not observed with the Hfx. volcanii DHS. Furthermore, the growth of Hfx. volcanii was not inhibited by the classical DHS inhibitor GC7. We propose a model of deoxyhypusine synthesis in Hfx. volcanii that differs from the canonical eukaryotic pathway, paving the way for further studies.

Molecular Factors of Hypochlorite Tolerance in the Hypersaline Archaeon Haloferax volcanii

“…In thermophiles, differences in polyamine ratios are correlated with growth temperature [ 52 ]. H. volcanii produces the polyamines agmatine and cadaverine [ 53 , 54 ] and has two SpeE homologs (HVO_B0357 and HVO_0255) that are predicted to be integral membrane proteins with conserved active site residues and structural homology to polyamine aminopropyltransferases ( Figure S9 ). Interestingly, the H. volcanii SpeEs were found to differ in primary sequence at key regions known to alter polyamine binding specificity in thermophilic polyamine aminopropyltransferases [ 55 ]; HVO_0255 had a GG(GA)G(F/Y) motif and long C-terminal extension, while HVO_B0357 had a GGGD(W/Y) motif and short-C-terminal tail ( Figures S9 and S10 ).…”

Polyamine function in archaea and bacteria

“…Halophiles do not accumulate either spermidine or putrescine; however, they do accumulate agmatine. An agmatinase-like gene (agmatinase converts agmatine to putrescine) is necessary for deoxyhypusine formation in Haloferax volcanii, and only deoxyhypusine, and not hypusine, is detected in aIF5A (26). The H. volcanii agmatinase-like gene is essential for growth even though putrescine and spermidine are not accumulated.…”

“…2). Although spermidine is required for deoxyhypusine formation in T. kodakarensis (26), putrescine is not required for growth because spermidine is synthesized from agmatine via aminopropylagmatine rather than putrescine (27). Some methanogens, in particular the Methanosarcinaceae, accumulate only homospermidine rather than spermidine (13).…”

“…An interesting question then is whether polyamines have been lost from halophiles because they are not capable of performing their usual functions in high KCl or whether high KCl renders the noncovalent function of polyamines superfluous. Certainly, halophiles have evolved to supply an aminobutyl group for deoxyhypusine formation without the participation of spermidine or homospermidine (26).…”

Modification of translation factor aIF5A from Sulfolobus solfataricus

“…Since so far only homologues of the DHS enzyme have been identified but no homologs of the second enzyme, DOHH, the archaeal hypusination pathway remains unsolved. A partial characterization of the protein has been carried out in S. acidocaldarius (Bartig et al 1992 ) and, more recently, in H. volcanii (Prunetti et al 2016 ). The latter contains only deoxyhypusinylated aIF5A, whose synthesis differs from the canonical eukaryotic pathway: in the first reaction, the DHS enzyme transfers agmatine to the aIF5A lysine, while in the second reaction the agmatinase enzyme leads to production of deoxyhypusine.…”