title: Dual biosynthesis pathway for longer-chain polyamines in the hyperthermophilic archaeon Thermococcus kodakarensis.
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 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 homogeneity, 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-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 kcat/Km value for agmatine than for putrescine, suggesting that TK0147 functions primarily as an aminopropyl transferase to produce N1-aminopropylagmatine. In T. kodakarensis, spermidine is produced mainly from agmatine via N1-aminopropylagmatine. Furthermore, spermine and N4-aminopropylspermine were detected in the TK0147 disruptant, indicating that TK0147 does not function to produce spermine and long-chain polyamines.
authors.affiliation: Department of Bioscience, School of Science and Technology, Kwansei-Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan.
authors.forename: Nanako
authors.initials: N
authors.lastname: Morimoto
authors.forename: Wakao
authors.initials: W
authors.lastname: Fukuda
authors.forename: Nanami
authors.initials: N
authors.lastname: Nakajima
authors.forename: Takeaki
authors.initials: T
authors.lastname: Masuda
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Terui
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Kanai
authors.forename: Tairo
authors.initials: T
authors.lastname: Oshima
authors.forename: Tadayuki
authors.initials: T
authors.lastname: Imanaka
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Fujiwara
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D011073:Polyamines / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D019712:Thermococcus / Q000235:genetics / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D011073:Polyamines