The function of the hypusine-containing proteins of yeast and other eukaryotes is well conserved

Magdolen, Viktor; Klier, Hannelore; Wöhl, Thorsten; Klink, Friedrich; Hirt, Heribert; Hauber, Joachim; Lottspeich, Friedrich

doi:10.1007/bf00282755

Cited by 42 publications

(39 citation statements)

References 21 publications

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“…It has already been shown that the eIF5A precursor protein isolated from a plant (i.e. alfalfa) can be activated by hypusine formation in yeast (35). The sequence of tobacco deoxyhypusine synthase shares all strictly conserved residues found in the primary structure of the enzymes from other sources (Fig.…”

Section: Resultsmentioning

confidence: 94%

Deoxyhypusine Synthase from Tobacco

Ober

Hartmann

1999

Journal of Biological Chemistry

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Deoxyhypusine synthase catalyzes the formation of a deoxyhypusine residue in the translation eukaryotic initiation factor 5A (eIF5A) precursor protein by transferring an aminobutyl moiety from spermidine onto a conserved lysine residue within the eIF5A polypeptide chain. This reaction commences the activation of the initiation factor in fungi and vertebrates. A mechanistically identical reaction is known in the biosynthetic pathway leading to pyrrolizidine alkaloids in plants. Deoxyhypusine synthase from tobacco was cloned and expressed in active form in Escherichia coli. It catalyzes the formation of a deoxyhypusine residue in the tobacco eIF5A substrate as shown by gas chromatography coupled with a mass spectrometer. The enzyme also accepts free putrescine as the aminobutyl acceptor, instead of lysine bound in the eIF5A polypeptide chain, yielding homospermidine. Conversely, it accepts homospermidine instead of spermidine as the aminobutyl donor, whereby the reactions with putrescine and homospermidine proceed at the same rate as those involving the authentic substrates. The conversion of deoxyhypusine synthase-catalyzed eIF5A deoxyhypusinylation pinpoints a function for spermidine in plant metabolism. Furthermore, and quite unexpectedly, the substrate spectrum of deoxyhypusine synthase hints at a biochemical basis behind the sparse and skew occurrence of both homospermidine and its pyrrolizidine derivatives across distantly related plant taxa.The eukaryotic initiation factor 5A (eIF5A), 1 a small 17.4-kDa protein, is activated by a post-translational modification of a specific lysine residue to hypusine (N ⑀ -(4-amino-2-hydroxybutyl)lysine) in an enzyme-catalyzed two-step mechanism (reviewed in Refs. 1 and 2). In the first step, the aminobutyl moiety of the polyamine spermidine is transferred by deoxyhypusine synthase (EC 1.1.1.249) in an NAD ϩ -dependent reaction to the ⑀-amino group of a specific lysine residue in the eIF5A precursor protein to form deoxyhypusine. In the second step, deoxyhypusine hydroxylase (EC 1.14.99.29) catalyzes the hydroxylation of the deoxyhypusine residue to hypusine. Activated eIF5A is the only protein in which the unusual amino acid hypusine has been detected to date (3, 4), the modification is one of the most specific post-translational modifications known (5, 6). eIF5A seems to be ubiquitous among eukaryotes (7) and archaebacteria (8). Its amino acid sequence is highly conserved, and the 12 amino acids surrounding the hypusine residue are identical in all eukaryotes studied. Although known for nearly 2 decades, the function of eIF5A is still obscure. Because of its in vitro activity in stimulating methionyl puromycin synthesis (9), eIF5A was classified as a protein synthesis initiation factor, though subsequent doubts have arisen as to whether initiation of protein synthesis is a major function of this protein (2). Using a yeast mutant, it was shown that depletion of eIF5A causes an immediate inhibition of cell growth but only a moderate inhibition (30%) of total protein synthe...

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Section: Resultsmentioning

confidence: 94%

Deoxyhypusine Synthase from Tobacco

Ober

Hartmann

1999

Journal of Biological Chemistry

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“…In the present EF-P crystal structure, the Arg-32 side chain protrudes toward the solvent at the end of the domain I arm. The bacterial EF-P reportedly lacks hypusine (13). Nevertheless, the conservation of the amino acid residues in the ␤3-␤4 connective linker and the basic residue at the tip of the loop implies the significance of this region for EF-P function.…”

Section: Structure Comparison Of Ef-p With Trna and Ribosome-binding mentioning

confidence: 99%

“…The enzyme that modifies lysine to hypusine in eIF-5A is essential for yeast viability (12). On the other hand, hypusine is not found in bacteria (13). The structures of eIF-5A from three Archaea, Methanococcus jannaschii, Pyrobaculum aerophilum, and Pyrococcus horikoshii, have two domains, which are composed of several ␤-strands (14)(15)(16).…”

mentioning

confidence: 99%

Crystal structure of elongation factor P from Thermus thermophilus HB8

Hanawa-Suetsugu

Sekine

Sakai

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

105

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Translation elongation factor P (EF-P) stimulates ribosomal peptidyltransferase activity. EF-P is conserved in bacteria and is essential for cell viability. Eukarya and Archaea have an EF-P homologue, eukaryotic initiation factor 5A (eIF-5A). In the present study, we determined the crystal structure of EF-P from Thermus thermophilus HB8 at a 1.65-Å resolution. EF-P consists of three ␤-barrel domains (I, II, and III), whereas eIF-5A has only two domains (N and C domains). Domain I of EF-P is topologically the same as the N domain of eIF-5A. On the other hand, EF-P domains II and III share the same topology as that of the eIF-5A C domain, indicating that domains II and III arose by duplication. Intriguingly, the N-terminal half of domain II and the C-terminal half of domain III of EF-P have sequence homologies to the N-and C-terminal halves, respectively, of the eIF-5A C domain. The three domains of EF-P are arranged in an ''L'' shape, with 65-and 53-Å-long arms at an angle of 95°, which is reminiscent of tRNA. Furthermore, most of the EF-P protein surface is negatively charged. Therefore, EF-P mimics the tRNA shape but uses domain topologies different from those of the known tRNA-mimicry translation factors. Domain I of EF-P has a conserved positive charge at its tip, like the eIF-5A N domain.

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“…Even though the precise in vivo function of this protein is still unknown, hypusine formation in eIF-5A was shown to be central to cell viability [1][2][3].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the metal ion requirement of the human deoxyhypusine hydroxylase from HeLa cells using a novel enzyme assay

Csonga¹,

Ettmayer²,

Auer³

et al. 1996

FEBS Letters

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Hypusine synthesis in the eukaryotic initiation factor 5A is a unique two-step posttranslational modification. After deoxyhypusine is generated by the deoxyhypusine synthase, the deoxyhypusine hydroxylase (EC 1.14.99.29) catalyzes the formation of mature hypusine. A rapid assay for monitoring the deoxyhypusine hydroxylase activity was established, employing the oxidative cleavage of the hypusyl residue and subsequent extraction of the generated aldehydes. As metal ion chelators have been reported to inhibit the deoxyhypusine hydroxylase, the mechanism of this inhibition and the effect of transition metal ions on the enzyme activity were investigated. A ferric ion appears to be essential for enzymatic activity, the inhibition of which is entirely attributed to the metal ion binding capacity of the chelators.

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The function of the hypusine-containing proteins of yeast and other eukaryotes is well conserved

Cited by 42 publications

References 21 publications

Deoxyhypusine Synthase from Tobacco

Deoxyhypusine Synthase from Tobacco

Crystal structure of elongation factor P from Thermus thermophilus HB8

Evaluation of the metal ion requirement of the human deoxyhypusine hydroxylase from HeLa cells using a novel enzyme assay

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