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.
TK0149 (designated as Tk-PdaD) of a hyperthermophilic archaeon, Thermococcus kodakaraensis, was annotated as pyruvoyl-dependent arginine decarboxylase, which catalyzes agmatine formation by the decarboxylation of arginine as the first step of polyamine biosynthesis. In order to investigate its physiological roles, Tk-PdaD was purified as a recombinant form, and its substrate dependency was examined using the candidate compounds arginine, ornithine and lysine. Tk-PdaD, expressed in Escherichia coli, was cleaved into alpha and beta subunits, as other pyruvoyl-dependent enzymes, and the resulting subunits formed an (alphabeta)6 complex. The Tk-PdaD complex catalyzed the decarboxylation of arginine but not that of ornithine and lysine. A gene disruptant lacking Tk-pdaD was constructed, showing that it grew only in the medium in the presence of agmatine but not in the absence of agmatine. The obtained results indicate that Tk-pdaD encodes a pyruvoyl-dependent arginine decarboxylase and that agmatine is essential for the cell growth of T. kodakaraensis.
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