A Collaborative Classroom Investigation of the Evolution of SABATH Methyltransferase Substrate Preference Shifts over 120 My of Flowering Plant History.

Next-generation sequencing has resulted in an explosion of available data, much of which remains unstudied in terms of biochemical function; yet, experimental characterization of these sequences has the potential to provide unprecedented insight into the evolution of enzyme activity. One way to make inroads into the experimental study of the voluminous data available is to engage students by integrating teaching and research in a college classroom such that eventually hundreds or thousands of enzymes may be characterized. In this study, we capitalize on this potential to focus on SABATH methyltransferase enzymes that have been shown to methylate the important plant hormone, salicylic acid (SA), to form methyl salicylate. We analyze data from 76 enzymes of flowering plant species in 23 orders and 41 families to investigate how widely conserved substrate preference is for SA methyltransferase orthologs. We find a high degree of conservation of substrate preference for SA over the structurally similar metabolite, benzoic acid, with recent switches that appear to be associated with gene duplication and at least three cases of functional compensation by paralogous enzymes. The presence of Met in active site position 150 is a useful predictor of SA methylation preference in SABATH methyltransferases but enzymes with other residues in the homologous position show the same substrate preference. Although our dense and systematic sampling of SABATH enzymes across angiosperms has revealed novel insights, this is merely the "tip of the iceberg" since thousands of sequences remain uncharacterized in this enzyme family alone.

Quantitation of Δ8-THC, Δ9-THC, Cannabidiol and 10 Other Cannabinoids and Metabolites in Oral Fluid by HPLC-MS-MS.

Quantitative analysis of Δ9-tetrahydrocannabinol (Δ9-THC) in oral fluid has gained increasing interest in clinical and forensic toxicology laboratories. New medicinal and/or recreational cannabinoid products require laboratories to distinguish different patterns of cannabinoid use. This study validated a high-performance liquid chromatography-tandem mass spectrometry method for 13 different cannabinoids, including (-)-trans-Δ8-tetrahydrocannabinol (Δ8-THC), (-)-trans-Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD), Δ9-tetrahydrocannabinolic acid-A (Δ9-THCA-A), cannabidiolic acid (CBDA), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-Δ9-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THCCOOH), tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), cannabidiorcol (CBD-C1), cannabichromene (CBC), cannabinol (CBN) and cannabigerol (CBG), in oral fluid. Baseline separation was achieved in the entire quantitation range between Δ9-THC and its isomer Δ8-THC. The quantitation range of Δ9-THC, Δ8-THC and CBD was from 0.1 to 800 ng/mL. Two hundred human subject oral fluid samples were analyzed with this method after solid phase extraction. Among the 200 human subject oral fluid samples, all 13 cannabinoid analytes were confirmed in at least one sample. Δ8-THC was confirmed in 11 samples, with or without the presence of Δ9-THC. A high concentration of 11-OH-Δ9-THC or Δ9-THCCOOH (>400 ng/mL) was confirmed in three samples. CBD, Δ9-THCA-A, THCV, CBN and CBG were confirmed in 74, 39, 44, 107 and 112 of the 179 confirmed Δ9-THC-positive samples, respectively. The quantitation of multiple cannabinoids and metabolites in oral fluid simultaneously provides valuable information for revealing cannabinoid consumption and interpreting cannabinoid-induced driving impairment.