The Onocerin story
Friday, 27 May 2016
It is always an interesting experience to close the door on a research project, although I confess to never believing that this can be done with any conviction, simply because the mind is never completely assured that everything has been achieved that could have been.
Post-doc work with a leader in one’s field is hopefully what many people aspire to, and I remember the first month when my boss (K E Bloch) suggested we eliminate NADP and oxygen as cofactor and participant in the cyclisation of squalene. The dogma had always been that all cyclised products derived their hydroxide groups from a reaction involving molecular oxygen and a reducing cofactor NADPH. We started with squalene epoxide which the neighbouring chemistry laboratory (E J Corey) had made in radioactive form and after making hog liver microsomes, We incubated the new substrate in the absence of oxygen and no added cofactors. The product (lanosterol) was the result of a magical enzyme (cyclase) which was produced in excellent yield.
The two groups went on to explore cyclises from plants such as β- amyrin and many years were spent attempting to purify these clever catalysts. To turn an essentially linear C-30 compound into a polycyclic structure with 8 optical centres, four and often five rings all joined in different ways was always going to be fascinating. In fact there are several thousand cyclic structures derived from this one reaction. These form the framework of a huge variety of useful plant products the properties of which are often poorly understood. Pharmacological research has focussed on the Chinese medical applications of plants which have been recorded for millennia in order to improve our understanding of this huge resource.
When I began my career as a lecturer, I chose to carry on working with cyclases and after coming across the only centrosymmetric triterpene (onocerin) which is found in a legume called Ononis, we decided to attempt to work out how the enzyme managed the synthesis. A cursory glance at the product suggests squalene is epoxidised at each end but the question has always been is the process sequential or not. Since no-one has isolated a half cyclised product except by genetically altering a monoepoxide cyclase, it was always assumed that the plant(s) managed to carry out the synthesis by some other method.
The product onocerin is made by a legume often found in sand dunes and golf courses. The English name is restharrow (the French call it arête boeuf, which is the same idea) basically making the farmer’s lives difficult on account of the huge tap root they presumably send down to recover a better water supply or something similar. It might of course be simply that the reason is because the plant dies back in the winter. One question we raised was “does Ononis use the lipid to slow the loss of water harvested from deep below the plant and can this property be exploited to serve crops in extremely dry areas?.” The converse property namely that of permitting mosses to survive in waterfalls and extreme damp conditions is explained by the property of this lipid, being a double ended structure, which might improve water tolerance; another useful trait which could have the added valuable resistance to fungal attack in crops. This idea could explain why species of water-loving plants such as club mosses, Selaginella make onocerin.
This is not the entire story, however. If we look at the global occurance of the onocerin framework, it has been found that Venezuelan oil deposits (and presumably other shale oil reservoirs) are full of compounds derived from onocerin, which probably means that dinosaur fodder was full of onocerin. Clearly the structure has been around for a long time. Another interesting occurrence of the compound is found in pine trees. Many pines (including the majority that cover Northern Labrador) make derivatives of onocerin called serratendiols. I have always assumed these are then converted into saponins which are likely candidates for fungicidal protection of the tree, similar to club mosses. Clearly we need to know more about this interesting cyclase and the products which depend on its activity. Can we modify the enzyme to make a whole new class of building blocks for the pharmaceutical and agrochemical industries? Are we looking at a whole new class of antifungals, not just to deal with rust in cereals, but aspergillus infections in humans? One group of Turkish scientists recently reported that onocerin and/or its saponins are ACE inhibitors. This exciting new property has neither been explained, nor exploited, at least as far as one has been able to discover.
This week the solution to the onocerin cyclase problem has been provided by a group from Meiji University in Japan. Cyclases have been studied for many years and a key observation is how well they are conserved. The Japanese team carried out PCR with primers common to many cyclases but chose to use the genes of Lycopodium (a club moss), and apart from finding sequences which were closely related to tetracyclic products like cycloartenol, they found two of which were unusual. These genes were cloned, put into a yeast which was modified to stop its ability to make lanosterol and therefore deficient in oxido squalene cyclase. The clones were quite different when challenged with the bis epoxide, LCC made a bicyclic product whilst LCD made nothing. The clever part of the experiment was to combine both the LCC and LCD genes in one yeast, when magically onocerin appeared as the only product. We now have a half cyclised product ‘pre-onocerin’ which never sees the light of day but is passes on to a second cyclase for ‘finishing’. This type of cooperative enzymology is rare in many of the enzymes we study and unique in cyclases, most or, dare I say, all of which have some very strange, highly conserved, features, including floppy chains (QW motifs), which help to link the quite beautiful array of 22 helices to each other to make spectacular twisted barrels in two structures one above the other. Interestingly the active site for many of the cyclases is a sequence DCTAE which is the key to a specific attack on the epoxide ring. What happens after this is a coordinated zipping up of the double bonds to form two, three, four and often five carbocyclic rings. Needless to say after 50 years with these enzymes, this week has been rather satisfying.
Cambio / Peter Dean