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

Abstract

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.

Polyamines in Eukaryotes, Bacteria, and Archaea

“…The aminopropyltransferase of Sulfolobus solfataricus was found to recognize a range of diamines and triamines (92). The variant AdoMet-dependent aminopropylagmatine pathway for Spd biosynthesis, previously identified in the bacterial extreme hyperthermophile T. thermophilus, was found in the euryarchaeote T. kodakaraensis (94).…”

Identification of a Novel Aminopropyltransferase Involved in the Synthesis of Branched-Chain Polyamines in Hyperthermophiles

“…Agmatine is also a precursor in the synthesis of agmatidine, an agmatine-conjugated cytidine found at the anticodon wobble position of archaeal tRNA Ile (33). Our genetic study revealed that TK0147 and TK0882 encode N 1 -aminopropylagmatine synthase and N 1 -aminopropylagmatine ureohydrolase, respectively, in the production of spermidine (20). Interestingly, larger quantities of agmatine accumulated in strain DAT, in which TK0147 is disrupted, than in the parental KU216 strain.…”

“…Spermidine and spermine are then produced by the addition of the aminopropyl group from decarboxylated Sadenosylmethionine (dcSAM). In contrast, thermophilic bacteria and archaea possess a unique polyamine biosynthetic pathway, in which spermidine is synthesized from agmatine via N 1 -aminopropylagmatine by aminopropyltransferase, followed by ureohydrolase (18,20,27).…”

“…One type consists of long linear polyamines such as caldopentamine [3333] NH, N,. Because the relative amounts of long/branched-chain polyamines in cells of (hyper)thermophiles were found to increase as growth temperatures increased, these unique polyamines are regarded as supporting the growth of thermophilic microorganisms under high-temperature conditions (18)(19)(20). An in vitro study indicated that long-chain and branched-chain polyamines effectively stabilized DNA and RNA, respectively (21), suggesting that these unique polyamines enhance translation efficiency under hightemperature conditions (22,23).…”

“…Spermidine and spermine are then produced by the addition of the aminopropyl group from decarboxylated Sadenosylmethionine (dcSAM). In contrast, thermophilic bacteria and archaea possess a unique polyamine biosynthetic pathway, in which spermidine is synthesized from agmatine via N 1 -aminopropylagmatine by aminopropyltransferase, followed by ureohydrolase (18,20,27).A sulfur-reducing hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, grows at temperatures between 60 and 100°C (28)(29)(30)(31). Our previous study found that T. kodakarensis PdaD (TK0149) catalyzed the synthesis of agmatine, the first step in polyamine biosynthesis, and was essential for cell growth (32).…”