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Elusive enzymes with biocatalytic potential: chlorinases in the plant kingdom

Periodic Reporting for period 1 - CHLOROIRIDOIDS (Elusive enzymes with biocatalytic potential: chlorinases in the plant kingdom)

Reporting period: 2015-12-01 to 2017-11-30

Plants produce a vast number of structurally diverse chemicals that act on us as dangerous toxins, vitamins, or medicinal compounds. Enzymes are the biological tools used for the production of such natural products and especially those in plants are underexplored since their genes are concealed by an unusual genetic complexity. The motivation for studying the biosynthesis of plant natural products is twofold. First, the biochemical secrets hidden in the blueberries that we eat for breakfast, in the snapdragon flowers planted in our garden and in the periwinkle leaves from which cancer drugs are extracted, deserve some curiosity. Second, the enzymes performing the synthesis of a natural product can serve chemists when they are repurposed as biocatalyts in “green” chemical reactions, shuffled together in synthetic biology platforms, or even reengineered for novel reactions.

Iridoids are a natural product class present in blueberries, snapdragon, Madagascar periwinkle and thousands of other plants. Only recently, the first steps of iridoid biosynthesis have been discovered. Especially a biosynthetic step discovered in Madagascar periwinkle in the O’Connor lab, leading from a linear precursor molecule to the characteristic bicyclic core structure of iridoids, has attracted considerable attention. This step is performed by the enzyme iridoid synthase. Later in the biosynthesis, the bicyclic core is rearranged and decorated with sugars, acids or other functional groups until, in many cases, the biosynthetic origin is hardly recognizable.

We noticed unusual features in the biosynthesis of iridoids in some plants that deserved further investigation. Previous publications on the biosynthesis of some iridoids suggested a noticeable structural variation of the core scaffold. Compared to the periwinkle iridoids, the configuration of one carbon atom is attached to the opposite side of the molecule. This seemingly small structural difference indicated an iridoid synthase with opposite stereospecificity. We identified the “epi-iridoid synthase” performing this reaction and sought to understand the molecular origin of the inverted stereospecificity. Investigations of the reaction mechanism and comparisons of related iridoid synthases have challenged previous hypotheses about the function of iridoid synthases. Furthermore, some of these iridoids are converted to chlorinated derivatives. Since chlorine incorporating enzymes are generally rare in the plant kingdom and have been elusive in higher plants, we also searched for this enzyme, albeit not successfully.
We based our search for the epi-iridoid synthase on the assumption that this enzyme would be structurally related to the known iridoid synthase from periwinkle. Enzymes are encoded as genes in the genetic information of an organism. A similarity search indeed revealed four relatives of the iridoid synthase in the plant of interest. We cloned all genes and transferred them to a laboratory strain of the bacterium Escherichia coli in order to apply a standard procedure to obtain all candidate enzymes in pure form. After adding the linear precursor obtained by chemical synthesis, the purified candidate enzymes were tested for their ability to perform the iridoid synthase reaction.

Although all four candidates made at least traces of cyclized iridoids, only one showed a level of activity comparable to that of the periwinkle enzyme. We believe that this candidate is the epi-iridoid synthase. Our initial hypothesis predicted that the epi-iridoid synthase would not make the cyclic product identical to the compound present in periwinkle, but a stereoisomer differing in the spatial arrangement of atoms. However, when we first analyzed the product mixtures of the new enzyme, we did not observe any difference. Since our analytical method (GC-MS) was blind to the very subtle differences between exact “mirror images” (enantiomers) of compounds, there was still the possibility that both enzymes made not identical, but enantiomeric products. Indeed, a more sensitive analytical method (chiral GC-MS) was able to demonstrate the minuscule differences in physico-chemical properties caused by the opposite “handedness” of the products.

During the period supported by the Marie Skłodowska-Curie fellowship, results of this work have been presented at the NCCR Symposium on Chemical Biology in Geneva (poster) and at the CCBIO Symposium Industrial Biocatalysis in Zurich (invited talk). After the end of the fellowship, the fellow has given talks about this work at the Junior Researcher Symposium “Bioorganic Chemistry” in Jena and at the Biotechnology 2020 symposium, also in Jena. A manuscript about discovery and characterization of the epi-iridoid synthase is in preparation and scheduled for publication in early 2017. Furthermore, the fellow has authored reviews about “Biocatalysts from alkaloid producing plants” (Current Opinion in Chemical Biology, 2016), which is directly related to this work, and about “Biosynthetic engineering of nonribosomal peptide synthetases” (Journal of Peptide Science, 2016).
From a comparison of the iridoid synthase and epi-iridoid synthase reactions, we could conclude that the cyclization is probably triggered in the active site with high stereospecificity but then proceeds outside the enzyme where it is not tightly controlled anymore as we thought previously. This mechanistic scenario delineates the scope for biocatalytic applications of iridoid synthase. We now have an enzyme with the opposite stereospecificity to the previously known one, which may be useful for synthetic applications, but modifications of the enzyme can never lead to different outcomes of the cyclization reaction. Instead, interesting biocatalytic applications may be possible with alternative substrates. Furthermore, the epi-iridoid synthase may explain the biosynthesis of hundreds of natural products and the enzyme may become a reference point for the biosynthetic elucidation of these epi-iridoids. Iridoid synthase and epi-iridoid synthase seem to have very specific differences in the binding pocket responsible for the different functions of the enzymes. Therefore, we will probably be able to predict for unknown iridoid synthases only from sequence whether they make one or the other type of product.