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J. Biol. Chem., Vol. 277, Issue 40, 37098-37104, October 4, 2002
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From the Biotechnology Research Center, Toyama Prefectural
University, Toyama 939-0398, Japan, the Eubacterial diterpene cyclase genes had
previously been cloned from a diterpenoid antibiotic terpentecin
producer (Dairi, T., Hamano, Y., Kuzuyama, T., Itoh, N., Furihata, K.,
and Seto, H. (2001) J. Bacteriol. 183, 6085-6094). Their
products, open reading frame 11 (ORF11) and ORF12, were essential for
the conversion of geranylgeranyl diphosphate (GGDP) into terpentetriene
(TTE) that had the same basic skeleton as terpentecin. In this study, functional analyses of these two enzymes were performed by using purified recombinant enzymes. The ORF11 product converted GGDP into a
cyclized intermediate, terpentedienol diphosphate (TDP), which was then
transformed into TTE by the ORF12 product. Interestingly, the ORF12
product directly catalyzed the conversion of GGDP into three olefinic
compounds. Moreover, the ORF12 product utilized farnesyl
diphosphate as a substrate to give three olefinic compounds, which had the same structures as those formed from GGDP with the exception of the chain lengths. These results suggested that the ORF11
product with a DXDD motif converted GGDP into TDP by
a protonation-initiated cyclization and that the ORF12 product with a
DDXXD motif completed the transformation of TDP to the
olefin, terpentetriene by an ionization-initiated reaction followed by
deprotonation. The kinetics of the ORF12 product indicated that
the affinity for TDP and GGDP were higher than that of farnesyl
diphosphate and that the relative activity of the reaction converting
TDP into TTE was highest among the reactions using TDP, GGDP, or
farnesyl diphosphate as the substrate. These results suggested that an
actual reaction catalyzed by the ORF12 was the conversion of TDP into
TTE in vivo.
Functional Analysis of Eubacterial Diterpene Cyclases Responsible
for Biosynthesis of a Diterpene Antibiotic, Terpentecin*
,
Institute of
Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku
113-0032, Japan, the § Division of Agriculture and
Agricultural Life Science, The University of Tokyo, Bunkyo-ku 113-0032, Japan, and the ¶ Faculty of Applied Bioscience, Tokyo University
of Agriculture, Setagaya-ku 156-8502, Japan
*
This work was supported in part by a grant-in-aid
for scientific research from Japan Society for the Promotion of
Science.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Biotechnology
Research Center, Toyama Prefectural University, Toyama 939-0398, Japan. Tel.: 81-766-56-7500; Fax: 81-766-56-2498; E-mail:
dairi@pu-toyama.ac.jp.
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