Functiona l Analysis of Eubacterial Diterpene Cyclases Responsible for Biosynthesis of a Diterpene Antibiotic, Terpentecin

Abstract

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.

Studies on Biosynthetic Genes and Enzymes of Isoprenoids Produced by Actinomycetes

“…We named this new product as terpentedienol diphosphate (TDP, 2), based on structural similarities to TTE (3) and the presence of two double bonds and a diphosphate. We also confirmed that TDP (2) was converted into TTE (3) by Cyc2, showing that TDP (2) is the real intermediate converted from GGDP (1) by Cyc1 [76].…”

“…Under this reaction condition, the formation of TTE (3) was confirmed [76]. On the other hand, TTE (3) was not formed by the enzyme reaction in which Cyc2 was used first, followed by inactivation and the addition of Cyc1.…”

Comparison of the Enzymatic Properties ofent-Copalyl Diphosphate Synthases in the Biosynthesis of Phytoalexins and Gibberellins in Rice

“…GGDP was purchased from Sigma-Aldrich and further purified by column chromatography to remove such impurities as Mg 2þ . 25) All other chemicals were of analytical grade. The plasmid, pGEX-4T3 {N-terminal glutathione S-transferase-(GST) fused, GE Healthcare}, was used for expressing the recombinant enzymes.…”

A Modular Approach for Facile Biosynthesis of Labdane-Related Diterpenes

“…In addition, the three pGGxC constructs reported here will be useful for characterization of novel class I labdane-related diterpene synthases. Finally, we expect that a similar approach will provide access to labdane-related diterpenes and diterpenoid natural products derived from other class II intermediates such as clerodadienyl diphosphate (i.e., by incorporating class II diterpene cyclases that produce such compounds 15 into pGG), which will further broaden the reach of the modular approach to labdane-related diterpene biosynthesis demonstrated here. (5) a E. coli containing the indicated plasmid(s) produce the indicated labdane-related diterpene, as identified by GC-MS based comparison to authentic standards, with production levels ranging from 5 to 100 µg diterpene/L mixed phase culture (see Supporting Information).…”