Enzymatic C–C-Coupling Prenylation: Bioinformatics – Modelling
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340 CHIMIA 2009, 63, No. 6 BIOTRANSFORMATIONS doi:10.2533/chimia.2009.340 Chimia 63 (2009) 340–344 © Schweizerische Chemische Gesellschaft Enzymatic C–C-Coupling Prenylation: Bioinformatics – Modelling – Mechanism – Protein-Redesign – Biocatalytic Application Ludger Wessjohann*, Svetlana Zakharova, Diana Schulze, Julia Kufka, Roman Weber, Lars Bräuer, and Wolfgang Brandt* Abstract: The functional role of isoprenoids and especially enzymatic prenylation in nature and human application is briefly covered, with the focus on bioinformatical, mechanistical and structural aspects of prenyltransferases and terpene synthases. These enzymes are as yet underrepresented but perspectively useful biocatalysts for C–C couplings of aromatic and isoprenoid substrates. Some examples of the successful use in chemoenzymatic synthesis are given including an application for the otherwise difficult synthesis of Kuhistanol A. Computational structure-based site-directed mutagenesis can be used for rational enzyme redesign to obtain altered substrate and product specificities, which is demonstrated for terpene cyclases. Keywords: Aromatic prenyltransferases · Biocatalysis · Protein-redesign · Substrate specificity · Terpene synthases 1. Introduction many pathways and important metabolites prenyl transferases. Aromatic prenyltrans- is given by Bouvier et al.[4] All organisms ferases catalyse the formation of C–C Prenyl-converting or -transferring en- possess essential isoprenoids. The evolu- bonds between an aromatic substrate (usu- zymes are responsible for the formation tion of the immense metabolic diversity ally a phenol) and an isoprenoid diphos- or modification of naturally occurring originating from equally diverse isoprenoid phate as electrophile (see e.g. Scheme 2 for isoprenoids (including terpenoids and converting enzymes is not yet completely the reaction of UbiA-enzyme). Isoprenyl meroterpenoids). More than 50,000 dif- understood on the molecular and structural pyrophosphate synthases are responsible ferent compounds have been isolated from level. for enzymatic connections (C–C coupling) fungi, animals, microbial and predomi- Prenyl-converting enzymes are clas- of two prenyl moieties (either by head to nantly plant resources and thus represent sified into terpene synthases (cyclases), head or head to tail condensation) leading the most diverse family of natural prod- transferases and hydrolases/isomerases. to the elongation of the prenyl chain. In the ucts, of which approximately one half are Transferases can be subdivided in aro- case of protein prenyltransferases the pre- terpenoids (including steroids), the other matic prenyltransferases, isoprenylpyro- nyl cation moiety is usually transferred to half are chimeric compounds of moieties phosphate synthases, and protein prenyl- the side chain of a C-terminal cysteine by from other biosynthetic origins coupled transferases (Scheme 1), and other prenyl- formation of a C–S bond. to isoprenoids (meroterpenoids).[1–3] converting enzymes not assigned to one of An excellent overview and summary of these groups (e.g. geranylgeranyl hydroge- 1.1 Importance of Prenylated nase or squalene epoxidase). For all these Metabolites in Nature and Human enzymes, the catalysis mechanism starts Use with the cleavage of a pyrophosphate leav- Different types of terpenoids serve a ing group from a prenyl moiety to form an number of important physiological and so- intermediate allylic prenyl cation (Scheme cietal functions. Plant terpenoids are classi- 1, step I). Terpene synthases (cyclases) use fied as primary and secondary metabolites. this reactive intermediate to perform an Isoprenoids as part of the primary metabo- intramolecular electrophilic addition to a lism serve basic functions like cell growth C=C double bond to form a terpinyl (e.g. modulation and plant elongation. Primary for monoterpene synthases) or other cyclic metabolic terpenoids such as steroids are cation intermediate to produce a wide va- essential for the membrane function and riety of terpenoids e.g. limonene, pinene, act as hormones and bile acids. Carote- *Correspondence: Prof. Dr. L. Wessjohann; sabinene, camphene, and many others noids are responsible for light harvesting PD Dr. W. Brandt (Scheme 1, step IIa). and photoprotection, and ubiquinone, me- Leibniz Institute of Plant Biochemistry Prenyltransferases require a second nu- naquinone and plastoquinone are involved Department of Bioorganic Chemistry [1–3,5] Weinberg 3 cleophilic substrate to which the intermedi- in the electron transport. Secondary D-06120 Halle (Saale), Germany ate isoprenyl cation is transferred (Scheme metabolic terpenoids are involved in e.g. Tel.: LW: +49 345 5582 1301 1, step IIb). They comprise – among others defense or communication. Their human WB: +49 345 5582 1360 E-mail: [email protected] – oligoprenyl pyrophosphate synthases, applications include pharmaceuticals, fla- [email protected] protein prenyltransferases, and aromatic vours, fragrances, food supplements, such BIOTRANSFORMATIONS CHIMIA 2009, 63, No. 6 341 of plant secondary metabolism. All these enzymes contain an aspartate-rich motif in their sequence required to bind divalent metal ions, which are necessary for cata- lytic activity. All members of this class catalyse C-prenylations, i.e. they transfer the prenyl residue to a carbon atom of an aromatic substrate. Until now, no X-ray structure is available for this group, but a first homology model could be developed by us recently.[25] The second group of aromatic prenyl- transferases[26] consists of soluble enzymes with no sequence similarity to the enzymes of the first class. So far, four members have been biochemically characterised: CloQ,[27,28] involved in clorobiocin biosyn- thesis, NphB[29] (naphterpin biosynthesis), Fnq26[30,31] (furanonaphthochinon I bio- synthesis) and SCO7190.[29] None of these enzymes contains aspartate-rich motifs, though the activity of NphB depends on Mg2+.[29] The experimental structure of the latter revealed a novel barrel architecture, the ABBA fold. This refers to a cylindri- cal β-sheet consisting of ten antiparallel β-strands surrounding a solvent-filled core. Four times a sequence of two β-strands fol- lows two -helices resulting in a ( ) Scheme 1. Classification of enzymes and related general mechanism of prenylations. Except from α ααββ 4 protein prenyltransferases and hydrolases/isomerases, the three other enzyme families catalyse structure. The remaining β-strands are ap- C–C-coupling prenylation. H–T = head to tail, H–H head to head condensation (e.g. squalene pended in a (αββα) structure, to which synthase with additional removal of OPP, product not shown), PP/OPP = pyrophosphate. the term ABBA refers. Because of high sequence similarities, this fold is assumed to be characteristic for the enzymes of this second class. In contrast to the aromatic as vitamins or sweeteners. A wide range overview about diverse prenyl-transferring prenyltransferases mentioned before, of terpenoids have shown pharmaceutical enzymes. Fnq26 was observed to catalyse an allylic activity against human diseases such as (often referred to as ‘reverse’) prenylation, cancer.[6,7] The most prominent example considering the fact that C(3) rather than is paclitaxel, commonly known under the 2. Prenyl Transfer to C(1) (regular prenylation) of the prenyl registered trademark Taxol®. Taxol is a Aromatic Systems – Aromatic residue is connected to the aromatic sub- diterpenoid-derived anticancer drug from Prenyltransferases strate. All enzymes of the ABBA family the bark of the pacific yew tree Taxus brev- add the prenyl chain to a carbon atom of ifolia.[8] Another example is the sesquiter- 2.1 Bioinformatics the aromatic structure resulting in a C–C penoid arteminisin from Artemisia annua Aromatic prenyltransferases form a coupling. But Fnq26 and NphB are addi- which possesses anti-malaria activity.[9] heterogenic cluster of enzymes that trans- tionally able to catalyse O-prenylations, Also monoterpene rich essential oils, e.g. fer prenyl residues to C, O, or N atoms of whereby an oxygen atom of the aromatic from species of Mentha, with (–)-menthol, aromatic structures. Often this leads to an structure serves as prenyl acceptor. The are important products for the pharmaceu- increased bioactivity compared to the non- better structural knowledge and their eas- tical, agrochemical, food, flavour and fra- prenylated compounds, most due to an in- ier accessibility makes this class interest- grance industries.[10,11] In addition to their creased affinity to biological membranes.[14] ing for future chemoenzymatic synthesis pharmaceutical use, they can act as attrac- The enzymes are widespread. They can be of bioactive molecules.[32] tants, repellents, toxins or antibiotics, or as found in plants, bacteria, and fungi. In re- The third class consists of the soluble precursors to such bioactive compounds. cent years, significant progress was made fungal indol prenyltransferases.[33] Similar They are important for the interaction of concerning genetics and biochemistry of to the described ABBA prenyltransferases sessile plants with other organisms in the these enzymes yielding in the finding that they do not contain aspartate-rich motifs context of reproduction, defence, or sym- not just one type of aromatic prenyltrans- and none of them depend on the presence biosis.[12] ferases exists. Today three different classes of Mg2+. So far, enzymes involved in three Terpenoids are derived from the pre- are known that are mainly distinguished