Now, Takaaki Mitsuhashi, Makoto Fujita and colleagues discovered a functional terpene synthase in a giant virus. With the help of bioinformatics tools, three potential terpene synthases were identified, and it was shown that they are phylogenetically distinct from previously described terpene synthases. The researchers decided to focus on the one that contains the conserved DDXXD motif of class I terpene synthases and termed this candidate enzyme Orpheovirus IHUMI-LCC2 terpene synthase (OILTS). The enzyme was heterologously expressed in Escherichia coli, purified and its crystal structure solved at 1.45-Å resolution. The analysis revealed that OILTS consists mainly of α helices and that, considering its compact size of 278 amino acid residues, it has a relatively large cavity of 554 Å3 providing sufficient space to engage with substrate molecules. Mutational studies confirmed the importance of conserved residues for enzymatic activity and indicated a similar mechanism for substrate recognition as those reported for class I terpene synthases from cellular organisms. In vitro reactions showed that OILTS can cyclize (2E,6E)-farnesyl diphosphate (FPP, 1) to (+)-germacrene D-4-ol (2) (pictured) and that it is catalytically active between pH 4.5−10.5 and 4−50 °C. Activity is retained even after storage for 10 months at 4 °C. The catalytic efficiency of the enzyme was determined to be 5.18 × 104 M−1 s−1 with kcat = 4.35 s−1 and KM = 84.09 μM, which is comparable to the kinetic parameters of terpene synthases from cellular organisms.
The researchers could not identify genes in the virus that encode enzymes to tailor terpenoid products, nor enzymes that are able to produce the substrate (1) for the terpene synthetase. As such, it remains unclear whether and, if so, why this species is producing terpenoid natural products. Further studies in this direction might provide clues into the nature and evolution of giant viruses.
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