Diterpene Resin Acids and Olefins in Calabrian Pine (Pinus nigra subsp. laricio (Poiret) Maire) Oleoresin: GC-MS Profiling of Major Diterpenoids in Different Plant Organs, Molecular Identification and Expression Analysis of Diterpene Synthase Genes

A quali-quantitative analysis of diterpenoid composition in tissues obtained from different organs of Pinus nigra subsp. laricio (Poiret) Maire (Calabrian pine) was carried out. Diterpene resin acids were the most abundant diterpenoids across all the examined tissues. The same nine diterpene resin acids were always found, with the abietane type prevailing on the pimarane type, although their quantitative distribution was found to be remarkably tissue-specific. The scrutiny of the available literature revealed species specificity as well. A phylogeny-based approach allowed us to isolate four cDNAs coding for diterpene synthases in Calabrian pine, each of which belonging to one of the four groups into which the d3 clade of the plants’ terpene synthases family can be divided. The deduced amino acid sequences allowed predicting that both monofunctional and bifunctional diterpene synthases are involved in the biosynthesis of diterpene resin acids in Calabrian pine. Transcript profiling revealed differential expression across the different tissues and was found to be consistent with the corresponding diterpenoid profiles. The isolation of the complete genomic sequences and the determination of their exon/intron structures allowed us to place the diterpene synthase genes from Calabrian pine on the background of current ideas on the functional evolution of diterpene synthases in Gymnosperms.

Targeting active site residues and structural anchoring positions in terpene synthases

The sesterterpene synthase SmTS1 from Streptomyces mobaraensis contains several unusual residues in positions that are otherwise highly conserved. Site-directed mutagenesis experiments for these residues are reported that showed different effects, resulting in some cases in an improved catalytic activity, but in other cases in a loss of enzyme function. For other enzyme variants a functional switch was observed, turning SmTS1 from a sesterterpene into a diterpene synthase. This article gives rational explanations for these findings that may generally allow for protein engineering of other terpene synthases to improve their catalytic efficiency or to change their functions.

The Introduction of Hydrazone, Hydrazide, or Azepane Moieties to the Triterpenoid Core Enhances an Activity Against M. tuberculosis

Background: Triterpenoids exhibit a wide spectrum of antimicrobial activity. Objective: The objective of this study was to synthesize a series of nitrogen derivatives based on lupane, oleanane, and ursane triterpenoids with high antitubercular activity. Methods: Isonicotinoylhydrazones were prepared via the reaction of 3-oxotriterpenic acids or betulonic aldehyde with isoniazid (INH) in yields of 54-72%. N-Acylation of betulonic or azepanobetulinic acids led to lupane C28 hydrazides and dihydrazides. The derivatives were evaluated for their in vitro antimycobacterial activities against Mycobacterium tuberculosis (MTB) H37RV and single-drug resistance (SDR)-TB in the National Institute of Allergy and Infectious Diseases, USA. Molecular docking was performed to evaluate the possible binding modes of investigated compounds in the active site of Diterpene synthase (Rv3378c). Results: The obtained compounds are represented by C3 or C28 conjugates with hydrazine hydrate or INH. Some compounds demonstrated from high minimum inhibitory concentration (MIC ≤ 10 μg/mL) to excellent (MICs from 0.19 to 1.25 μg/mL) activity against MTB H37RV. Two lupane conjugates with INH were the leading compounds against MTB H37RV and some SDR-strains with MICs ranged from 0.19 to 1.70 μg/mL. Molecular docking of active compounds to diterpene synthase showed that these moieties accommodate the active site of the enzyme. Conclusions: It was revealed that the conjugation of lupanes with INH at C3 is more effective than at C28 and the lupane skeleton is preferable among oleanane and ursane types. The replacement of native hexacarbocyclic A ring to seven-member azepane ring is favorably for inhibition of both MTB H37RV and SDR-strains. These data could possibly mean that the antitubercular activity against INH-resistant strains (INH-R) came from both triterpenoid and isoniazid parts of the hybrid molecules. Azepanobetulin showed the highest activity against both INH-R strains in comparison with other triterpenoids and INH. Thus, the introduction of hydrazone, hydrazide (dihydrazide), or azepane moieties into the triterpenoid core is a promising way for the development of new anti-tubercular agents.