Deoxyhypusine synthase from tobacco. cDNA isolation, characterization, and bacterial expression of an enzyme with extended substrate specificity.
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.
Ober
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Institut für Pharmazeutische Biologie der Technischen Universität Braunschweig, Mendelssohnstrasse 1, D-38106 Braunschweig, Germany.
Hartmann
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D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D010947:Plants, Toxic; D016415:Sequence Alignment; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology*
D018076:DNA, Complementary; D000587:Oxidoreductases Acting on CH-NH Group Donors; C063507:deoxyhypusine synthase; D019883:Alkyl and Aryl Transferases; C028376:homospermidine synthetase