A stable intermediate product of the archaeal zinc-containing 7Fe ferredoxin from Sulfolobus sp. strain 7 by artificial oxidative conversion

AbstractIrreversible conversion of the purified zinc-containing 7Fe ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7 was found to occur under aerobic conditions at pH 5.0 and at 4°C. This process accompanied a substantial increase of the electron paramagnetic resonance signal attributed to a [3Fe-4S]1+ cluster, and the converted form containing ∼6 Fe/Zn (mol/mol) had a net charge different from that of the native 7Fe ferredoxin. These data provide evidence for the formation of a stable intermediate product of the archaeal ferredoxin having two [3Fe-4S] clusters and a zinc center by artificial oxidative degradation. This further explains the discrepancy that a zinc center and two [3Fe-4S] clusters (but not a zinc center and one [3Fe-4S] cluster plus one [4Fe-4S] cluster) are observed in the crystal structure at pH 5.0

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

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) fromS. 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

Domain Motion in 3-isopropylmalate dehydrogenase: A Strategy to Enhance its Thermal Stability

In order to elucidate the thermal properties of Thermus thermophilus 3-isopropylmalate dehydrogenase, mutant structures with mutations at the C-terminus were compared with each other. The structural movement can be anticipated from the structural changes among mutants in regions of a minor groove and pillar. Our previous studies revealed that the open-close movement of the active site groove antagonizes to that of the minor groove (like a paperclip) and the thermostability of the enzyme increases when the active site groove is closed. In the present study, it is shown that the motion of the enzyme mainly occurs in the first domain and strand D in the pillar structure is a hinge-bending region of the movement. The motion of the first domain to expand the minor groove may close the active site groove suggesting a mechanism for the enhanced thermal stability of 3-isopropylmalate dehydrogenase

Domain Motion in 3-isopropylmalate dehydrogenase: A Strategy to Enhance its Thermal Stability

In order to elucidate the thermal properties of Thermus thermophilus 3-isopropylmalate dehydrogenase, mutant structures with mutations at the C-terminus were compared with each other. The structural movement can be anticipated from the structural changes among mutants in regions of a minor groove and pillar. Our previous studies revealed that the open-close movement of the active site groove antagonizes to that of the minor groove (like a paperclip) and the thermostability of the enzyme increases when the active site groove is closed. In the present study, it is shown that the motion of the enzyme mainly occurs in the first domain and strand D in the pillar structure is a hinge-bending region of the movement. The motion of the first domain to expand the minor groove may close the active site groove suggesting a mechanism for the enhanced thermal stability of 3-isopropylmalate dehydrogenase