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

Abstract

ABSTRACT 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°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°C and 93°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°C and 93°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.