Flowers of Nicotiana species emit a characteristic blend including the cineole cassette monoterpenes. This set of terpenes is synthesized by multiproduct enzymes, with either 1,8-cineole or a-terpineol contributing most to the volatile spectrum, thus referring to cineole or terpineol synthase, respectively. To understand the molecular and structural requirements of the enzymes that favor the biochemical formation of a-terpineol and 1,8-cineole, site-directed mutagenesis, in silico modeling, and semiempiric calculations were performed. Our results indicate the formation of a-terpineol by a nucleophilic attack of water. During this attack, the a-terpinyl cation is stabilized by p-stacking with a tryptophan side chain (tryptophan-253). The hypothesized catalytic mechanism of a-terpineol-to-1,8-cineole conversion is initiated by a catalytic dyad (histidine-502 and glutamate-249), acting as a base, and a threonine (threonine-278) providing the subsequent rearrangement from terpineol to cineol by catalyzing the autoprotonation of (S)-(2)-a-terpineol, which is the favored enantiomer product of the recombinant enzymes. Furthermore, by site-directed mutagenesis, we were able to identify amino acids at positions 147, 148, and 266 that determine the different terpineol-cineole ratios in Nicotiana suaveolens cineole synthase and Nicotiana langsdorffii terpineol synthase. Since amino acid 266 is more than 10 Å away from the active site, an indirect effect of this amino acid exchange on the catalysis is discussed.
The previously introduced ratio of frequencies (RF) framework provides statistically sound information on the relative interaction preferences of atoms in crystal structures. By applying the methodology to protein‐ligand complexes, we can investigate the significance of interactions that are employed in structure‐based drug design. Here, we revisit three aspects of molecular recognition in the light of the RF framework, namely stacking interactions of heteroaromatic rings with protein amide groups, interactions of acidified C−H groups, and interaction differences between syn and anti lone pairs of carboxylate groups. In addition, we introduce a highly interactive visualization tool that facilitates design idea generation in structure‐enabled drug discovery projects. Finally, we show that applying the RF analysis as a simple rescoring tool after docking improves enrichment factors for the DUD−E diverse targets subset supporting the relevance of our approach.
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