While this method strongly benefits from the established handling of the fast-growing E.
coli cells and the quick headspace analysis, there are still some disadvantages for this approach to consider. Firstly, a GC/MS analysis can only identify known TS products by their EI mass spectra and retention indices. Secondly, not every TS product is suitable for headspace analysis, which may lead to false impressions of TS reactivity depending on the volatility of its product(s). Moreover, non-engineered E. coli as a host has some limitations regarding precursor availability. For instance, since there is no GGPPS known from E. coli, diterpene synthase activity may be overlooked. This case is also true for the discussed TS from C. pinensis, which showed DTS activity for in vitro incubations with GGPP being the subject of the introduced publication.
Summary
Interestingly, sesquiterpene synthase (e. g. 53) activity was not observed in vitro for the purified, recombinant TS from C. pinensis. Instead, a clean conversion of GGPP to the diterpene alcohol 18-hydroxydolabella-3,7-diene (201, Figure 19) was found.[235] The compound was isolated and characterised by NMR, while its absolute configuration was deduced from incubation experiments with (R)- and (S)-(1-13C,1-2H)GGPP complemented with the GGPPS mediated elongation of (R)- and (S)-(1-13C,1-2H)GPP with IPP followed by conversion to labelled 201.
Figure 19. Structure of the DTS product 18-hydroxydolabella-3,7-diene (202).
The DTS was also expressed in N. benthamiana, which resulted in the production of 201 with a yield of 0.03% of fresh leaf weight underlining the ability of plants to serve as a sustainable production platform for terpenes.[229] To exclude experimental errors, the headspace analysis of E. coli expressing the TS was repeated to also observe 200 by GC/MS analysis. The detailed reasons for the production of 53 in E. coli remain unknown, despite the different conditions of the reaction in this host compared that of the in vitro incubation may lead to a reasonable explanation. Therefore, care should be taken when working with heterologous expression systems for they might not reflect the actual biochemical situation in the TS’ native host. Since none of the discussed products have been found in C. pinensis cultures, also the in vitro investigations perhaps face the same problems. Overall, this study encourages the continuing work on characterised TSs for interesting aspects of their reactivities may have been overlooked.
Chapter 10
Spata-13,17-diene Synthase—An Enzyme with Sesqui-, Di-, and Sesterterpene Synthase Activity from Streptomyces xinghaiensis
Jan Rinkel, Lukas Lauterbach and Prof. Dr. Jeroen S. Dickschat*
Angew. Chem. Int. Ed. 2017, 56, 16385–16389; Angew. Chem. 2017, 129, 16603–16607.
Reprinted from Angew. Chem. Int. Ed. 2017, 56, 16385–16389 and Angew. Chem. 2017, 129, 16603–16607 with kind permission from John Wiley and Sons.
My contributions to this work included cloning and heterologous expression of the DTS, isolation and NMR characterisation of its products, incubation experiments with isotopically labelled substrates and analysis of results together with site-directed mutagenesis and metal-ion dependency studies.
The publication “Spata-13,17-diene Synthase—An Enzyme with Sesqui-, Di-, and Sesterterpene Synthase Activity from Streptomyces xinghaiensis” is attached in Appendix I.
Introduction
The majority of TSs only accept one oligoprenyl diphosphate substrate of a defined chain length. This substrate selectivity is explainable by the evolutionary tailoring of the active site architecture, which needs to provide a defined cavity for the cyclisation that usually does not possess the flexibility to cyclise longer or shorter substrates in the same way.
However, in plants more promiscuous substrate conversion is occasionally observed, as exemplified by -selinene synthase and -humulene synthase from Abies grandis producing both cyclised sesquiterpenes from FPP and cyclised monoterpenes from GPP.[236] Several other examples were also reported.[237] This promiscuity may also be beneficial for the host organism, because only one enzyme can produce terpenes of different classes depending on the biochemical environment such as precursor availability in different cell compartments. Expanding the substrate scope of terpene synthases also raises the question whether extreme chain lengths are still convertible by a TS. For instance, the biosynthesis of the unusual tetraterpene poduran (202, Figure 20), which was isolated from the springtail Podura aquatica,[238] is completely unknown, still, there might be one TS responsible for the cyclisation of the partially saturated or unsaturated long chain substrate.
Figure 20. Structure of the tetraterpene poduran (202) from Podura aquatica.[238] The relative configurations of the methyl branches are unknown.
Another way to biochemically change the substrate or product scope of a TS are different metal ions. While TSs are usually Mg2+-dependent, also Mn2+ occasionally works as a good substituent. This was demonstrated with a related PT from the beetle Phaedon cochleariae, which produces mainly FPP (82%) in the presence of Mg2+, whereas the selectivity changes towards GPP (96%), if Co2+ or Mn2+ are present. This mechanism may also be of ecological relevance to switch between mono- and sesquiterpene production.[37]
In this publication, a DTS from the marine actinomycete Streptomyces xinghaiensis is characterised, which features a surprisingly broad substrate scope by converting FPP, GGPP and GFPP. The cyclisation mechanism was investigated by labelling experiments, the metal ion dependency of the TS was addressed and several structurally important amino acid residues were changed by site-directed mutagenesis.
Summary
A TS from S. xinghaiensis was characterised as a spata-13,17-diene (208, Scheme 27) synthase (SpS).[239] This diterpene hydrocarbon features a prenyl side chain and a tricyclic core structure with a four-membered ring. As a side product of the conversion with GGPP (33), also prenylkelsoene (212) could be isolated, which possesses an altered core structure. The conversion of GFPP (34) gave the sesterterpene homologues 209 and 213, from which only 209 was isolated whereas 213 was tentatively assigned based on its similar EI-MS spectrum to 212. A conversion of FPP did not yield the sesquiterpene core structures. Instead, germacrene A (53) was isolated. Combining these results, a cyclisation mechanism was proposed, which is depicted in Scheme 27.
After a 1,10Re-cyclisation, cations 203 are further converted by deprotonation to build up the cyclopropane ring neutral intermediates 204 and 205. The known compound cneorubin Y (204)[240] could also be isolated from the reaction with GGPP. A reprotonation at C-3 induces further 2Si,6Si-cyclisation to cations 206, which are the central branching points towards the two different core structures. With an opening of the three-membered ring, either a 1,7Si-ring closure leads to 207 (path a) giving rise to the main products 208 and 209 after deprotonation, or a 7Si,10-cyclisation yields 211 (path b), which in turn after deprotonation gives the side products 212 and 213. The sesquiterpene cation 203 may also be deprotonated to yield 53 in case of FPP.
Scheme 27. Proposed cyclisation mechanism of SpS[239] and the originally reported structure of bourbon-11-ene (prespatane, 215). For simplicity, multiple structures are occasionally included in one structure number.
This mechanism was supported by conversion of the twenty isotopomers of (13C1)GGPP to yield labelled diterpenes in the expected positions. The stereochemical course of the
and the corresponding GPP and FPP isotopomers were used to assign the absolute configurations. Interestingly, the NMR data of the sesquiterpene core structure of 208 matched very well with the reported chemical shifts of the sesquiterpene bourbon-11-ene (prespatane, 215),[241] suggesting that the reported different relative configuration of this compound needs revision. This would also better explain the biosynthetic relationship of 215 to kelsoene[241] (tritomarene) in a similar way as suggested for SpS.
Indeed, the same structural revision was also suggested from crystallographic data in a different report, which was published at the same time.[242] To investigate the tolerance of SpS towards a saturated side chain as present in a longer version in 202, 10,11-dihydro-FPP was elongated with IPP by GGPPS to 14,15-dihydro-GGPP, whose incubation with SpS yielded 210. Also the kelsoene derivative 214 could be observed by GC/MS. Therefore, similar enzymes might also be involved in the biosynthesis of 202.
Besides the well-known conserved motifs of TSs as discussed in Chapter 1.1.4, a sequence alignment of 51 bacterial TSs also revealed several other similarities on the amino acid level. This observation is not surprising, since the overall low sequence conservation compared to the highly similar secondary and tertiary structures of TSs can only be explained by certain structure inducing conserved residues. This was investigated for SpS by site-directed mutagenesis identifying P83, L90, E184 and E217 (which is changed in native SpS to D) to be of structural importance. A comparison of the corresponding positions in the crystal structure of selinadiene synthase[64f] lead to their proposed functions as helix turns, part of Mg2+ binding and part of a salt bridge between different helices. Also different metal ions were tested with Mg2+ and Mn2+
promoting activity. Whereas the conversion with Mn2+ was even higher for the wild type enzyme, the D217E mutant showed no activity with Mn2+ suggesting a role of the corresponding salt bridge for the size of the metal binding site.
Overall, the ability of SpS to catalyse similar reactions on different substrate lengths is remarkable and raises the question, how this reactivity is accomplished by the architecture of the active site, which may contain a hydrophobic flexible tunnel to take up different linear isoprenoid side chains.