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Characterization of a novel linalool-metabolizing enzyme in Castellaniella defragrans 65 Phen


Scilipoti,  Stefano
IMPRS MarMic, Max Planck Institute for Marine Microbiology, Max Planck Society;

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Scilipoti, S. (2016). Characterization of a novel linalool-metabolizing enzyme in Castellaniella defragrans 65 Phen. Master Thesis, Universität Triest, Triest, Italien.

Cite as: https://hdl.handle.net/21.11116/0000-0001-C370-4
Isoprenoids, also known as terpenoids, are naturally occurring hydrocarbons. To date, more than 60000 different compounds have been discovered, mainly due to a great potential for enzymatic derivatization starting from a common scaffold. Monoterpenes constitute a class of terpenoids with ten carbon atoms synthesized mainly by plants and microorganisms. They possess a peculiar scent and their properties including antimicrobial, anti-herbivore, antioxidant and antitumor find a plethora of applications, for instance in cosmetic, food and pharmaceutical industries. The monoterpene biosynthesis involves the presence of a class of enzymes called terpene synthases, which are widely distributed in plant, fungal and bacterial species. Among them, terpene cyclases catalyze the transformation of an acyclic pyrophosphate into a cyclic compound. Below toxic concentrations, bacteria can use monoterpenes as source of carbon and energy. Under anaerobic and denitrifying conditions, the betaproteobacterium Castellaniella defragrans 65 Phen is capable of metabolizing monoterpenes as sole carbon and energy source. The linalool dehydratase/isomerase is an enzyme with a crucial role in the metabolism of acyclic monoterpenes in C. defragrans, catalyzing the hydration of myrcene to linalool and the isomerization of linalool to geraniol. The deletion mutant C. defragrans 65 Phen Δldi failed to grow on myrcene or geraniol, in contrast to the wildtype. But the mutant strain grew on linalool, suggesting the presence of a hitherto unknown enzyme utilizing linalool. In this study, the metabolic fate of linalool was explored. C. defragrans 65 Phen Δldi was grown on linalool and nitrate in a multiple-fed batch fermentation. In cell-free protein extracts, the formation of two monocyclic monoterpenes, α-terpinene and terpinolene, was detected. The transformation required ATP – likely for the activation of the substrate - and divalent cations. The novel enzyme was shown to be a soluble protein. It did not require other low molecular weight cofactors and was stable after 24 hours at 4°C. The novel enzyme was partly purified by size exclusion chromatography and ion exchange chromatography. Size exclusion chromatography revealed a molecular weight of 200 kDa for the enzyme. The optimal incubation time for the partly-purified protein was shown to be 4 hours. During this time the enzyme activity was unchanged. Furthermore, the magnesium-manganese dependence was characterized. The enzyme achieved a much higher activity when just magnesium Mg2+ was supplemented as metal cofactor and ATP as a cosubstrate. The suggested mechanism starts with the activation of linalool to linalyl-pyrophosphate as intermediate, proceeds with the cyclization to the α-terpinyl carbocation and ends with a deprotonation leading to the two final products, α-terpinene and terpinolene. In summary, the enzyme activity was partially purified, the apparent molecular weight was determined and magnesium ions as sole cofactor were identified. This will enable in the future a purification via several chromatographies to yield highly pure enzyme, hopefully few protein bands in the SDS PAGE gel. These proteins may be analyzed by matrix assisted laser desorption/ionization – time of flight mass spectroscopy (MALDI-ToF). Characterizing the oligopeptide sequence of the protein, this technique will identify the gene(s) corresponding to the active protein bands. The genes can be subsequently overexpressed. This information can be also used to compare obtained sequence with those already available in public databases and will confirm the novelty of this putative monoterpene cyclase. The so-far-obtained insights on the chemical characterization and from the purification not only enrich our knowledge of the partially characterized novel enzyme, but also confirm that many monoterpene synthases work better in presence of magnesium only as divalent cofactor. Additional modeling through X-ray crystallography or small angle X-ray Scattering (SAXS) will provide us insights on the structure and on the potential interactions with unknown cofactors, substrates and metal cations.