Purification of deoxyhypusine synthase from Neurospora crassa to homogeneity by substrate elution affinity chromatography.
Deoxyhypusine synthase is an NAD(+)-dependent enzyme that catalyzes the formation of deoxyhypusine residue on the eIF-5A precursor by using spermidine as the substrate. Deoxyhypusine synthase bound tightly to 1,12-diaminododecane-agarose and could be eluted selectively by spermidine. This finding enabled us to develop a simple two-column procedure to purify deoxyhypusine synthase from Neurospora crassa to apparent homogeneity. The purified enzyme had a specific activity of 130,000 units/mg of protein, representing a 64,000-fold purification from cell extracts. Size exclusion chromatography indicated that the native enzyme had a molecular mass of 180 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the pure enzyme showed a single band at the 40-kDa position, suggesting that Neurospora deoxyhypusine synthase is a homotetramer. Deoxyhypusine synthase appeared to be hydrophobic and required non-ionic detergent such as Tween 20 to stabilize the activity. Treatment of the enzyme with sulfhydryl reagents resulted in a complete loss of activity. Inclusion of NAD+ reduced the inactivation rate by manyfold, indicating the presence of -SH groups at or near the active site. Partial amino acid sequences of four peptide fragments that cover about one quarter of the enzyme were obtained for cDNA and genomic cloning work.
Tao
Y
Y
Department of Chemistry, Rutgers, State University of New Jersey, Piscataway 08855-0939.
Chen
K Y
KY
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology*; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D013439:Sulfhydryl Reagents; D000587:Oxidoreductases Acting on CH-NH Group Donors; C063507:deoxyhypusine synthase