Plant compounds make up many of the derived medicines, poisons, and generally useful molecules used in the world today and for much of human history. While we may have learned to produce several of them synthetically as technology has advanced, the original knowledge on their properties and how to use them came from nature. Even today, we may rely on growing certain plants for particular compounds due to an inability to manufacture them any other way. When that happens, the main bottleneck in production is how many of said plants we can grow and how much of the compound they produce individually.

Biotechnology and genetics researchers have long considered such plant production pathways to be a potential gold mine if we can find out how they turn on and off their production and what the limiting steps are for the reactions. With that noted, terpenoids are among the most diverse group of chemicals made by plants and commonly serve in the wild as pathogen deterrents, especially for harmful insect species. Terpenoids at the same time are fundamental for plant growth and development, among other things, showcasing that diversity of function they employ.

How To Make Terpenoids

For human usage, these compounds can be found in fragrances, flavorings, insecticides, medicines as previously noted, and even biofuels. A big product from terpenoids is Taxol, a chemotherapy drug that works on five different major forms of cancer. One would think with all this complexity that the pathway to produce them would be similarly complicated with multiple odds and ends to make such a disparate collection of molecular structures. But it turns out to be quite the opposite.

The starting positions begin with two molecules, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which themselves can be both made by two other pathways depending on their location in the cell. These both are then elongated thanks to the work of enzymes known as farnesyl diphosphate synthases (FPPSs). How this generally works is that one molecule of DMAPP is combined with two molecules of IPP to synthesize the obviously named farnesyl diphosphate (FPP). The FPPs form the basis for multiple other types of terpene creation.

The rate limiting step that prevents massive terpenoid production is generally accepted to be the step done by the enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR). But when HMGR was overexpressed in past experiments, this was still unable to overcome the limitations, implying there are other factors involved in preventing the production of plants with high amounts of terpenoids.

A Variable Out Of Place

It is at this point that our study today comes into play, a collaboration between Purdue University and the Salk Institute. Their research had previously revealed that there is a third, additional pathway involved in terpenoid synthesis, where a different related molecule is made called isopentenyl phosphate kinase (IPK). This enzyme moves itself to the cytoplasm, where it begins transforming the basic forms of isopentenyl phosphate (IP) and, it is suspected, dimethylallyl phosphate (DMAP) to IPP and DMAPP. This is done via phosphorylation, hence the extra phosphate (P) in their names, though ATP is needed to provide the phosphate and energy for the reaction.

In bacteria, it is known that IPK is used to form an extra, essential step in the primary pathway that creates an alternate, split direction for the synthesis to go. These paths are used to make more IP for IPK to then turn into their more complex forms. But plants lack the relevant genes used to create IP in this manner. So the question is where does the IP come from in order for IPK, which is known to be in plants, to do its phosphorylation? So the hunt was on to find what the mystery culprit could be that filled in this missing hole in the alternative terpenoid pathway.

A recent publication had found a hydrolase of the Nudix superfamily that was able to dephosphorylate IPP back into IP, giving the possibility that other Nudix members might have related properties. The 27 Arabidopsis Nudix enzymes were expressed in E. coli and tested for being phosphatases. Then, among those, they were tested for having activity with IPP in particular. Only two, AtNudx1 and AtNudx3, were able to do the IPP to IP change. So, they were shown to have this activity in a lab tube, but what about in plants themselves?

Upon measuring the activity of both within Arabidopsis, both were found to be expressed in all tissues, though AtNudx3 produced far more mRNA than its counterpart. In depth measurements proved what had been already suspected, these Nudix hydrolases in particular appeared to be controlling the process of turning IPP into IP. This instantly showed the differences between bacteria and plants. It appears there is no direct synthesis of IP in plant systems, instead there is just recycled usage of IPP, with only some of it being dedicated for higher up terpenoid formation.

The Knowledge of Production

This explains why terpenoid levels are always so low in plant tissues. They have no way to produce more, as they have a limited and stagnant amount of IPP to work with and there are a lot of terpenoids and even other compounds that it is needed for. Further testing by transgenically adding in an IP production gene from bacteria into tobacco plants does seem to activate an alternative junction in the biological pathway, much like what is seen in said bacteria, but in tobacco plants instead.

Overexpressing this added gene and also an Arabidopsis HMGR gene, which was previously thought to be the rate limiting step of the entire system, created a synergistic effect that highly boosted the creation of certain kinds of terpenoids.

Figuring out that higher terpenoid concentrations can be induced in transgenic plants is a huge step toward future hopes of being able to produce vast amounts of the compounds for use in medicine and everything else. But we have to fully grasp the biochemical pathways involved before that will be possible and now we have a far wider knowledge of how terpenoids are produced than before. We might finally be at the point where we can put the production plans into action.

The significance this holds for certain cancer drugs and other important products cannot be overstated. Every little bit helps and being able to properly supply critical chemicals like these will end up saving thousands of lives.

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Photo CCs: Tolmukapea from Wikimedia Commons

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