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From structure to mechanism

Life would be impossible without the broad range of enzymes that enable the flow of cellular metabolites in plant, animal and microbial organisms. Enzymes are biocatalysts that control and facilitate difficult chemical reactions associated with inheritance, breathing and digestion as well as the synthesis of natural products. Enzymes like chorismatase have long been attractive drug discovery targets. Junior professor Dr. Jennifer Andexer from the Institute of Pharmaceutical Sciences at the University of Freiburg was involved in the discovery of a new chorismatase family and is now specifically focusing on the enzymes’ three-dimensional structure from where she deduces information on their function. In the long term, she also hopes to be able to construct building blocks for the biosynthesis of natural products.

Junior Prof. Dr. Jennifer Andexer’s research is focused on chorismatases. © Dr. Jennifer Andexer, University of Freiburg

Plants, fungi and bacteria use chorismic acid or chorismate, the anionic form of the acid, for producing aromatic amino acids such as phenylalanine, tyrosine and tryptophan. This pathway is not present in animals, and therefore not in humans either. Chorismate and chorismate-converting enzymes (i.e. chorismatases) are therefore ideal drug discovery targets.

While mutagenesis is the directed molecular modification of enzymes and is used for improving biocatalysts, mutasynthesis combines the power of synthetic biology and chemical synthesis to generate medicinally valuable natural products. The idea is to manipulate biosynthetic pathways in order to produce natural product derivatives with new cytotoxic and antibiotic properties and fewer adverse effects.

According to Dr. Jennifer Andexer from the Institute of Pharmaceutical Sciences at the University of Freiburg, researchers who want to apply rational design processes need to have detailed knowledge of the enzyme they would like to modify. Rational design is the creation of new molecules by using models to predict the effect of the molecules’ structure on their behaviour. Andexer’s project has been receiving funding from the Baden-Württemberg government since October 2013 and will be funded for a total of three years. During a research stay at the University of Cambridge, UK, Andexer was part of a team that discovered chorismatase enzymes, but has since tended to concentrate more on the enzymes’ characteristics and functions. Her colleagues from Cambridge are still very much focused on exploring the chorismatase biosynthesis pathways.

Small structural differences lead to different mechanisms of action

3D diagram of chorismatase FkbO. Green arrows: beta-sheet structure of the polypeptides, blue: alpha helices, red: active centre. © Dr. Jennifer Andexer, University of Freiburg

“We are taking individual enzymes out of the biosynthesis pathway and having a closer look at their structure,” said the biologist. “This is important because the three-dimensional structure of an enzyme is closely related to its mechanism of action.” Chorismatases are usually part of complex biosynthesis clusters involving numerous enzymes, whose subsequent action results in the generation of large molecules such as rapamycin or tacrolimus.

Chorismate (from Greek choris = to separate) plays an important role as a branch-point in the biosynthesis of aromatic amino acids as well as of many other cyclic compounds. All these compounds are synthesised in the shikimic acid pathway, a metabolic route that is used by bacteria, fungi and plants to produce essential amino acids as well as ubichinone, vitamin K and E, anthocyanins and salicylic acid. 

Andexer is aware of a broad range of enzymes that metabolise chorismate and is specifically interested in their function. “We are interested in how and why different products are synthesised from chorismate. We are also interested in how the various chorismatases differ from each other.” Andexer and her team have already isolated and identified two important chorismatase types, FkbO and Hyg5. They are classified according to the compounds (FkbO: 3,4-dihydroxycyclohexa-1,5-dienoic acid; Hyg5: 3-hydroxybenzoic acid) produced in addition to pyruvate. Andexer’s major research focus is on these two biocatalysts and she has already found out that the two enzymes have different three-dimensional structures and use different mechanisms for hydrolysing chorismate.

“In spite of these differences, the FkbO- and Hyg5-type chorismatase amino acid sequences are nevertheless extremely similar. We therefore want to find out why, in spite of this similarity, the two enzymes differ to such a large extent.” Andexer’s team has previously worked with another group of researchers in a project that used crystallographic methods to explore the three-dimensional structure of the FkbO enzyme type. We will now continue the laborious task of trying to find out more about the enzyme's mechanism of action. “We have looked at the FkbO structure and have come up with an idea as to how the enzyme might hydrolyse chorismate. We are now trying to produce experimental evidence for our hypothesis.”

Basic research in the foreground

Andexer’s major focus is on producing building blocks for specific synthesis pathways. The compound that results from the hydrolysis of chorismate (a transdiol) is of particular interest to the researchers due to its chiral structure. A molecule is chiral if it cannot be superposed onto its mirror image. The chemical production of chiral molecules is rather difficult and is only possible using a complex, multi-tier process. “FkbO chorismatases can be used to produce chiral molecules that can then be used for other syntheses,” said Andexer. “This enables us to simplify relatively complex chemical processes.”

Andexer’s team will spend the next three years trying to solve as yet unsolved questions. Further chorismatase types besides the aforementioned FkbO and Hyg5 types would appear to exist, and the scientists want to study the differences between them. They also want to study the relationship between chorismate lyases, synthases and mutases and FkbO and Hyg5 by comparing the structure of these enzymes and their mechanisms of action. There is also an isochorismatase which, although it uses isochorismate as natural substrate, uses the chorismatase mechanism of action for metabolising chorismate in a side reaction. The scientists would like to obtain detailed insights into the chorismate pathways and elucidate the relationships between the chorismate-dependent enzymes.

Chorismatase reaction.
The chorismatase types FkbO and Hyg5 metabolise chorismate, but generate completely different products. © Dr. Jennifer Andexer, University of Freiburg

Applications: rapalogues and tacrologues

Detailed knowledge of enzymes and their involvement in biosynthesis pathways is absolutely necessary for potential manipulations. Mutasynthesis couples chemical synthesis with molecular biology in order to develop medically effective products. Many immunosuppressive drugs like rapamycin and tacrolimus are produced with bacteria that use chorismate as a substrate. Knocking out certain reaction components and introducing modified building blocks produces rapamycin and tacrolimus analogues (i.e. rapalogues and tacrologues). These derivatives are more effective and can also be better tolerated by patients than the originals.

Further information:

Jun.-Prof. Dr. Jennifer Andexer
Institute of Pharmaceutical Sciences
University of Freiburg
Albertstr. 25 (Otto-Krayer-Haus)
79104 Freiburg
Tel.: +49 (0)761 / 203 - 67398
Fax: +49 80)761 / 203 - 6351
E-mail: jennifer.andexer(at)pharmazie.uni-freiburg.de
Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/from-structure-to-mechanism