Dual Biosynthesis Pathway for Longer-Chain Polyamines in the Hyperthermophilic Archaeon Thermococcus kodakarensis

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 N-4-aminopropylspermine [3(3) 43] were identified as major polyamines at 60 degrees C, and the amounts of N-4-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 degrees C and 93 degrees C, and the growth was partially retrieved by the addition of spermidine. In the TK0882 gene disruptant, agmatine and N-1-aminopropylagmatine accumulated in the cytoplasm. Recombinant TK0882 was purified to homogeneity, and its ureohydrolase characteristics were examined. It possessed a 43-fold-higher k(cat)/K-m value for N-1-aminopropylagmatine than for agmatine, suggesting that TK0882 functions mainly as N-1-aminopropylagmatine ureohydrolase to produce spermidine. TK0147, annotated as spermidine/spermine synthase, was also studied. The TK0147 gene disruptant showed a remarkable growth defect at 85 degrees C and 93 degrees C. Moreover, large amounts of agmatine but smaller amounts of putrescine accumulated in the disruptant. Purified recombinant TK0147 possessed a 78-fold-higher k(cat)/K-m value for agmatine than for putrescine, suggesting that TK0147 functions primarily as an aminopropyl transferase to produce N-1-aminopropylagmatine. In T. kodakarensis, spermidine is produced mainly from agmatine via N-1-aminopropylagmatine. Furthermore, spermine and N-4-aminopropylspermine were detected in the TK0147 disruptant, indicating that TK0147 does not function to produce spermine and long-chain polyamines.