Dual Biosynthesis Pathway for Longer-Chain Polyamines in the

Long-chain and/or branched-chain polyamines are unique polycations found in thermophiles. Cytoplasmic polyamines were analyzed for cells cultivated at various growth temperatures in the hyperthermophilic archaeon Thermococcus kodakarensis. Spermidine [34] and N4-aminopropylspermine [3(3)43] were identified as major polyamines at 60°C, and the amounts of N4-aminopropylspermine [3(3)43] increased as the growth temperature rose. To identify genes involved in polyamine biosynthesis, a gene disruption study was performed. The open reading frames (ORFs) TK0240, TK0474, and TK0882, annotated as agmatine ureohydrolase genes, were disrupted. Only the TK0882 gene disruptant showed a growth defect at 85°C and 93°C, and the growth was partially retrieved by the addition of spermidine. In the TK0882 gene disruptant, agmatine and N1-aminopropylagmatine accumulated in the cytoplasm. Recombinant TK0882 was purified to homoge-neity, and its ureohydrolase characteristics were examined. It possessed a 43-fold-higher kcat/Km value for N1-aminopropylagmatine than for agmatine, suggesting that TK0882 functions mainly as N1-aminopro