Conformational Analysis of (+)-Germacrene D-4-ol Using the Crystalline Sponge Method to Elucidate the Origin of its Instability.

Unsaturated cyclic terpenes often exhibit instability due to the proximation of C=C bonds in the cyclic skeleton, leading to nonenzymatic degradation. In this study, the crystalline sponge (CS) method was employed for the X-ray conformational analysis of a minute amount of oily and cyclic terpene compound, (+)-germacrene D-4-ol, which was produced by a terpene synthase OILTS under in vitro conditions.  The CS method revealed a reactive conformation of the cyclic terpene with proximal double bonds. Under weakly acidic in vivo conditions, OILTS gave four pseudo-natural products or artifacts. The CS method also elucidated the structures of these degraded compounds, proposing a degradation mechanism triggered by the transannular reactions.

Functional Plasticity of a Viral Terpene Synthase, OILTS, that Shows Non-Specific Metal Cofactor Binding and Metal-Dependent Biosynthesis.

OILTS is a viral class I terpene synthase found from the giant virus Orpheovirus IHUMI-LCC2. It exhibits a unique structure and demonstrates high plasticity to metal cofactors, allowing it to biosynthesize different cyclic terpene frameworks. Notably, while OILTS produces only (+)-germacrene D-4-ol with the most common cofactor, Mg2+, it also biosynthesizes a different cyclic terpene, (+)-cubebol, with Mn2+, Co2+, or Ni2+, presenting a rare instance of cofactor-dependent enzyme catalysis. This is the first report of (+)-cubebol biosynthesis, to our knowledge. In addition, OILTS can uptake Zn2+ as a cofactor, which is uncommon among ordinary terpene synthases. These findings suggest that OILTS's functional plasticity may benefit the virus in diverse host environments, highlighting potential evolutionary implications.

Function and Structure of a Terpene Synthase Encoded in a Giant Virus Genome.

Giant viruses are nonstandard viruses with large particles and genomes. While previous studies have shown that their genomes contain various sequences of interest, their genes related specifically to natural product biosynthesis remain unexplored. Here we analyze the function and structure of a terpene synthase encoded by the gene of a giant virus. The enzyme is phylogenetically separated from the terpene synthases of cellular organisms; however, heterologous gene expression revealed that it still functions as a terpene synthase and produces a cyclic terpene from a farnesyl diphosphate precursor. Crystallographic analysis revealed its protein structure, which is relatively compact but retains essential motifs of the terpene synthases. We thus suggest that like cellular organisms, giant viruses produce and utilize natural products for their ecological strategies.

Solvent Effects in the Crystalline Sponge Method: Importance of Co-solvents for Ordering Absorbed Guests.

In the crystalline sponge method, the crucial step for ordering the absorbed guest is soaking of the guest into the pores of the crystalline sponge. Here, we find that the choice of solvent is particularly important for smooth guest soaking and ordering. Moderately polar solvents, such as ketones and esters, which we have previously avoided for the guest-soaking process, efficiently promote diffusion and guest ordering by filling the gaps in the pores through co-crystallization with the guests. Using this modified protocol, we successfully demonstrate the structural analysis of various steroids.