N1-Aminopropylagmatine, a New Polyamine Produced as a Key
Intermediate in Polyamine Biosynthesis of an Extreme
Thermophile, Thermus thermophilus *
Received for publication, November 27, 2004, and in revised form, June 14, 2005
Published, JBC Papers in Press, June 27, 2005, DOI 10.1074/jbc.M413332200
Mio Ohnuma‡, Yusuke Terui‡, Masatada Tamakoshi‡, Hidemichi Mitome§, Masaru Niitsu¶,
Keijiro Samejima¶, Etsuko Kawashima§, and Tairo Oshima‡?
From the ‡Department of Molecular Biology and the §School of Pharmacy, Tokyo University of Pharmacy and Life
Science, 1432-1 Horinouchi, Hachioji, Tokyo, 192-0392 and the ¶Faculty of Pharmaceutical Sciences, Josai University,
1-1 Keyakidai, Sakado, Saitama 530-0248, Japan
In the extreme thermophile Thermus thermophilus, a
disruption mutant of a gene homologous to speB (coding
for agmatinase ? agmatine ureohydrolase) accumulated
azaoctane, N8-amidinospermidine), a new compound,
whereas all other polyamines produced by the wild-type
strain were absent from the cells. Double disruption of
speB and speE (polyamine aminopropyltransferase) re-
sulted in the disappearance of N1-aminopropylagmatine
and the accumulation of agmatine. These results sug-
gested the following. 1) N1-Aminopropylagmatine is pro-
duced from agmatine by the action of an enzyme coded
by speE. 2) N1-Aminopropylagmatine is a metabolic in-
termediate in the biosynthesis of unique polyamines
found in the thermophile. 3) N1-Aminopropylagmatine is
a substrate of the SpeB homolog. They further suggest a
new biosynthetic pathway in T. thermophilus, by which
polyamines are formed from agmatine via N1-aminopro-
pylagmatine. To confirm our speculation, we purified
the expression product of the speB homolog and con-
firmed that the enzyme hydrolyzes N1-aminopropylag-
matine to spermidine but does not act on agmatine.
Polyamines play important roles in cell proliferation and cell
differentiation. Common polyamines such as putrescine, sper-
midine, and spermine are distributed ubiquitously in cells and
tissues at relatively high concentrations (1, 2).
Thermus thermophilus, of which the genome project was
completed using two strains, HB8 and HB27 (Structural-Bio-
logical Whole Cell Project at www.srg.harima.riken.go.jp/
thermus/j_index.htm and see Ref. 3, respectively), produces a
variety of polyamines including unusually long polyamines and
branched ones (4) (see Fig. 9C). These long and branched poly-
amines have a marked effect of protecting and stabilizing nu-
cleic acids (5, 6) and of activating cell-free polypeptide synthe-
sis at high temperature (7–9).
In many organisms, such as bacteria, yeast, animals, and
plants, the first step of polyamine biosynthesis is production of
putrescine by decarboxylation of L-ornithine (see Fig. 9A). An
additional or alternative pathway of putrescine biosynthesis
that is often seen in plants and sometimes in bacteria is decar-
boxylation of L-arginine followed by hydrolysis of agmatine.
Agmatine ureohydrolase or agmatinase, coded by the speB
gene, catalyzes this second reaction. The next step is production
of spermidine and spermine by the addition of an aminopropyl
group to putrescine and spermidine, respectively. This reaction is
catalyzed by spermidine or spermine synthase (putrescine/sper-
midine aminopropyltransferase) coded by the speE gene (1).
To investigate the polyamine biosynthetic pathway in
T. thermophilus, we constructed a disruption strain of the speB
gene homolog of T. thermophilus. Disruption of the speB gene
homolog resulted in drastic reduction of triamines, longer and
branched polyamines without accumulation of agmatine, and
in accumulation of an unknown compound. Double disruption
of speB and speE gene homologs resulted in disappearance of
this compound and accumulation of agmatine in the cells. The
new compound was identified as N1-aminopropylagmatine (N8-
amidino-1,8-diamino-4-azaoctane, N8-amidinospermidine) by
comparison with the chemically synthesized authentic com-
pound. In vitro reactions revealed that SpeE is responsible for
the production of N1-aminopropylagmatine, and SpeB converts
N1-aminopropylagmatine to spermidine.
Strains and Culture Conditions
The strains of T. thermophilus and Escherichia coli and plasmids
used in this study are listed in Table I. The rich growth and minimum
media for T. thermophilus were as described previously (10). Leucine,
isoleucine, and uracil (50 ?g/ml each) were included in the minimum
medium. Media were solidified as described (11).
Construction of Plasmids
All nucleotide sequences of T. thermophilus HB8 used in this study
were kindly provided by Dr. Seiki Kuramitsu of Osaka University.
Construction of pSBKm and pSEPE is shown in Fig. 1. PCR was carried
out for 25 cycles (94 °C for 0.5 min, 55 °C for 0.5 min, and 72 °C for 1
min) with LA Taq in GC buffer (Takara Bio) using pairs of oligonucleo-
tide primers listed in Table II. Two pairs of primers, speBKpnUp5?-
speBHinUp3? and speBEcoDw5?-speBXbaDw3?, were used to construct
pSBKm; and an additional two pairs, speEup5?kpn-speEup3?hin and
speEdw5?xba-speEdw3?sac, were used to construct pESE. Genomic
DNA of T. thermophilus HB8 was used as the PCR template. PCR
products were digested with restriction endonucleases listed in Table II
and cloned into pBluescript SK?or pBHTK (14) to construct pSBUKm,
pSBD, pSEUKm, and pSED. The 3?-half of speB and the downstream
* This work was supported in part by Grants-in-aid for Scientific
Research 11794038 and 1665704 and by grants-in-aid for promoting
bioventures in private universities from the Ministry of Education,
Culture, Sports, Science and Technology, the Japanese Government.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
The atomic coordinates and structure factors (code 1UIR) have been
deposited in the Protein Data Bank, Research Collaboratory for Struc-
? To whom correspondence should be addressed: Dept. of Molecular
Biology, Tokyo University of Pharmacy and Life Science, 1432-1 Horin-
ouchi, Hachioji, Tokyo, 192-0392, Japan. Tel.: 81-426-76-7134; Fax:
81-426-76-7145; E-mail: firstname.lastname@example.org.
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 280, No. 34, Issue of August 26, pp. 30073–30082, 2005
Printed in U.S.A.
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region of speE were inserted into pSBUKm and pSEUKm to prepare
pSBKm and pSEKm, respectively. The HTK region of pSEKm was
replaced with the pyrE gene to construct pSEPE.
To make speE overexpression plasmid, PCR was carried out for 25
cycles of 98 °C for 0.5 min and 72 °C for 3 min with PfuTurbo DNA
polymerase (Stratagene) by using primers of sE5?NdeI and sE3?Hind.
To make speB overexpression plasmid, PCR was carried out for 25
cycles (94 °C for 0.5 min, 55 °C for 0.5 min, and 72 °C for 1 min) with LA
Taq in GC buffer using primers of sB5?NdeI and sB3?Hind (Table II).
Each PCR product was digested with NdeI and HindIII and was cloned
between the NdeI and HindIII site of expression plasmid pET21c? to
make pESE8 and pESB8, respectively.
All cloned nucleotide sequences were determined to verify the fidelity
of the amplified product to the original sequence using BigDye Termi-
nator Ready Reaction Premix (PerkinElmer Life Sciences) and an ABI
PRISM 377 DNA Sequencing System (PerkinElmer Life Sciences).
Bacterial strains and plasmids used in this study
Strain or DNA Description/genotypeSource or Ref.
Takara Bio (12)
T. thermophilus HB27 but ?leuB ?pyrE
T. thermophilus TTY1 but speB::HTK
T. thermophilus TTY1 but ?speE
T. thermophilus MOSE but speB::HTK
Plasmid carrying HTK gene
Plasmid carrying pyrE gene
Ampr, ?speE, HTK
Ampr, ?speE, pyrE?
Ampr, for speE overexpression
Ampr, for speB overexpression
FIG. 1. Construction of plasmids
used for gene disruption of speB and
speE. See “Experimental and Procedures”
for details. A, construction of pSBKm. B,
construction of pSEPE.
Aminopropylagmatine and a New Polyamine Biosynthetic Pathway
by guest on November 4, 2015
and Tairo Oshima
J. Biol. Chem.
Niitsu, Keijiro Samejima, Etsuko Kawashima
Tamakoshi, Hidemichi Mitome, Masaru
Mio Ohnuma, Yusuke Terui, Masatada
in Polyamine Biosynthesis of an Extreme
IntermediatePolyamine Produced as a Key
-Aminopropylagmatine, a New
Metabolism and Bioenergetics:
doi: 10.1074/jbc.M413332200 originally published online June 27, 2005
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