Taxing enzyme: Crystallography reveals biomolecular link

Ezine

Published: Jan 5, 2011
Author: David Bradley
Channels: X-ray Spectrometry

thumbnail image: Taxing enzyme: Crystallography reveals biomolecular link

Diversity beyond compare

The crystal structure of taxadiene synthase, an enzyme key to terpene biosynthesis in many living organisms, confirms a theoretically predicted link between two enzyme classes in the evolution of compounds such as the natural product anticancer drug Taxol.

Seemingly, the enzyme taxadiene synthase has a class I domain similar to that seen in another enzyme, pentalenene synthase and also class II regions in its protein structure that resemble those seen in yet another enzyme, squalene-hopene cyclase. Now, the first X-ray crystal structure of a diterpene cyclase provides an important clue as to why this should be the case. The findings will help open up this aspect of evolutionary history for the biosynthetic enzymes nature uses to make many complex natural products. It could also be exploited in engineering microbes, with mutants of the enzymes, that could be used to synthesise designer versions of such natural products for a range of applications in medicine, agrichemicals and elsewhere.

Mustafa Koeksal and David Christianson of the Department of Chemistry, at the University of Pennsylvania, Philadelphia, Yinghua Jin and Robert Coates of the University of Illinois at Urbana-Champaign, Urbana, Illinois, and Rodney Croteau of Washington State University, Pullman, Washington, USA, explain that there are some 55,000 terpenes and terpenoid natural products that science has so far identified.

Hydrocarbon construction

Terpenes are hydrocarbons constructed biochemically through the combination of several isoprene units. Terpenoids are modified terpenes, in which methyl groups have been moved or removed or oxygen atoms added. This incredibly diverse group of chemicals make up the scent of eucalyptus, minty plants, the flavours of cinnamon and cloves, the yellow colour of some flowers. In plants and animals terpenoids are the building blocks of vitamin A and steroidal hormones as well as being adjuncts to proteins that enhance cell membrane attachment or otherwise fine-tune functionality. They are not only fascinating from the point of view of understanding the evolution of life on earth at the chemical level, but many terpenoids have demonstrated medicinal activity. Among them the polycyclic diterpenoid Taxol (paclitaxel), from the Pacific yew tree (Taxus brevifolia), which is now known to promote tubulin polymerization in cells, which endows it with a notably potent anticancer activity in some forms of the disease.

Earlier research using X-ray diffraction has provided insights into how enzymatic control brings about the biosynthesis of terpenes and terpenoids, but the three-dimensional structure of a diterpene cyclase remained unknown until Christianson and colleagues tackled it. They reported in Nature the X-ray structure of taxadiene synthase, which catalyses the formation of the core molecular scaffold of Taxol.

Taxadiene synthase catalyses the cyclization of a linear terpenoid substrate geranylgeranyl diphosphate (GGPP) to make taxa-4(5),11(12)diene. The researchers knew that the full-form of the enzyme has 862 amino acid residues, of which an approximate 80-residue sequence is removed before the enzyme carries out its function.

As such, the team investigated a variant of the enzyme from which they had removed the transit and added a short chain at the N terminus to allow the structure to be determined more easily. They complexed this variant, which they refer to as TXS, with two substrates analogous to the natural target of the enzyme: 13-aza-13,14-dihydrocopalyl diphosphate (determined at 1.82 angstrom resolution) and 2-fluorogeranylgeranyl diphosphate (2.25 angstrom). The TXS structure reveals a definitive connection between the two distinct cyclase classes in the evolution of terpenoid biosynthesis, the team says.

Diversity applications

"Biosynthetic diversity in the family of terpenoid natural products is rooted in a 'mix and match' evolutionary strategy with class I and class II terpenoid cyclase folds, which can evolve together or separately as needed to generate the terpenoid product(s) required by the organism," the team explains. This simple solution to deriving greater diversity and complexity might now be used in laboratory-based biotransformation reactions or in generating modified bacteria for the "natural" synthesis of novel non-natural terpenoids with potential in medicine and other areas.

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.