Higher activity of recombinant bovine deoxyhypusine synthase vs. human deoxyhypusine synthase.
Mature eukaryotic initiation factor 5A (eIF5A) is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (Nepsilon-(4-amino-2(R)-hydroxybutyl)lysine). The synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Deoxyhypusine synthase is the first of the two enzymes that catalyzes the maturation of eIF5A. We have subcloned the cDNA encoding bovine and human deoxyhypusine synthase into a pET-11a expression vector, separately. T7-tagged bovine and human deoxyhypusine synthase have been overexpressed in Escherichia coli and purified to homogeneity using T7 antibody affinity chromatography. Activities of the enzyme from both human and bovine have been measured by their ability to convert the eIF5A precursor protein to the intermediate, deoxyhypusine form of eIF5A. Our results have shown that bovine deoxyhypusine synthase has considerably higher activity than human deoxyhypusine synthase in catalyzing the synthesis of deoxyhypusine.
Huang
Jenq-Kuen
JK
Department of Chemistry, Western Illinois University, One University Circle, Macomb, IL 61455, USA.
Tsai
Shuhui
S
Huang
George H
GH
Gowda
Prashanth G
PG
Walzer
Andrew M
AM
Wen
Lisa
L
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D000097574:Eukaryotic Translation Initiation Factor 5A
D008239:Lysine; D000587:Oxidoreductases Acting on CH-NH Group Donors; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A; C034924:deoxyhypusine; C063507:deoxyhypusine synthase