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J. Biol. Chem., Vol. 277, Issue 40, 37098-37104, October 4, 2002

This Article Full Text Full Text (PDF) All Versions of this Article: 277/40/37098    most recent M206382200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hamano, Y. Articles by Dairi, T. Search for Related Content PubMed PubMed Citation Articles by Hamano, Y. Articles by Dairi, T. Social Bookmarking                      What's this?

Functional Analysis of Eubacterial Diterpene Cyclases Responsible for Biosynthesis of a Diterpene Antibiotic, Terpentecin^*

Yoshimitsu Hamano, Tomohisa Kuzuyama, Nobuya Itoh, Kazuo Furihata^§, Haruo Seto^¶, and Tohru Dairi

From the Biotechnology Research Center, Toyama Prefectural University, Toyama 939-0398, Japan, the  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

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.

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