Structures of active-state orexin receptor 2 rationalize peptide and small-molecule agonist recognition and receptor activation.

Narcolepsy type 1 (NT1) is a chronic neurological disorder that impairs the brain's ability to control sleep-wake cycles. Current therapies are limited to the management of symptoms with modest effectiveness and substantial adverse effects. Agonists of the orexin receptor 2 (OX2R) have shown promise as novel therapeutics that directly target the pathophysiology of the disease. However, identification of drug-like OX2R agonists has proven difficult. Here we report cryo-electron microscopy structures of active-state OX2R bound to an endogenous peptide agonist and a small-molecule agonist. The extended carboxy-terminal segment of the peptide reaches into the core of OX2R to stabilize an active conformation, while the small-molecule agonist binds deep inside the orthosteric pocket, making similar key interactions. Comparison with antagonist-bound OX2R suggests a molecular mechanism that rationalizes both receptor activation and inhibition. Our results enable structure-based discovery of therapeutic orexin agonists for the treatment of NT1 and other hypersomnia disorders.

The identification of a novel lead class for phosphodiesterase 2 inhibition by fragment-based drug design.

We have identified a novel PDE2 inhibitor series using fragment-based screening. Pyrazolopyrimidine fragment 1, while possessing weak potency (Ki = 22.4 µM), exhibited good binding efficiencies (LBE = 0.49, LLE = 4.48) to serve as a start for structure-based drug design. With the assistance of molecular modeling and X-ray crystallography, this fragment was developed into a series of potent PDE2 inhibitors with good physicochemical properties. Compound 16, a PDE2 selective inhibitor, was identified that exhibited favorable rat pharmacokinetic properties.

Structural basis for the cooperative allosteric activation of the free fatty acid receptor GPR40.

Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40-MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.

Insights into activity and inhibition from the crystal structure of human O-GlcNAcase.

O-GlcNAc hydrolase (OGA) catalyzes removal of ßα-linked N-acetyl-D-glucosamine from serine and threonine residues. We report crystal structures of Homo sapiens OGA catalytic domain in apo and inhibited states, revealing a flexible dimer that displays three unique conformations and is characterized by subdomain α-helix swapping. These results identify new structural features of the substrate-binding groove adjacent to the catalytic site and open new opportunities for structural, mechanistic and drug discovery activities.

Integration of Affinity Selection-Mass Spectrometry and Functional Cell-Based Assays to Rapidly Triage Druggable Target Space within the NF-κB Pathway.

The primary objective of early drug discovery is to associate druggable target space with a desired phenotype. The inability to efficiently associate these often leads to failure early in the drug discovery process. In this proof-of-concept study, the most tractable starting points for drug discovery within the NF-κB pathway model system were identified by integrating affinity selection-mass spectrometry (AS-MS) with functional cellular assays. The AS-MS platform Automated Ligand Identification System (ALIS) was used to rapidly screen 15 NF-κB proteins in parallel against large-compound libraries. ALIS identified 382 target-selective compounds binding to 14 of the 15 proteins. Without any chemical optimization, 22 of the 382 target-selective compounds exhibited a cellular phenotype consistent with the respective target associated in ALIS. Further studies on structurally related compounds distinguished two chemical series that exhibited a preliminary structure-activity relationship and confirmed target-driven cellular activity to NF-κB1/p105 and TRAF5, respectively. These two series represent new drug discovery opportunities for chemical optimization. The results described herein demonstrate the power of combining ALIS with cell functional assays in a high-throughput, target-based approach to determine the most tractable drug discovery opportunities within a pathway.

Allergen stabilities and compatibilities in immunotherapy mixtures that contain cat, dog, dust mite, and cockroach extracts.

BACKGROUND: Indoor allergen mixtures that contain cat, dog, dust mite, and cockroach extracts are commonly used in allergy clinics for subcutaneous immunotherapy, but product-specific stabilities and mixing compatibilities in these complex patient formulas have not been determined. OBJECTIVES: To assess the recoveries of cat, dog epithelia, dog dander, dust mite Dermatophagoides farinae, and cockroach mix allergen activities in 5 component mixtures and 1:10 (vol/vol) dilutions stored for up to 12 months. METHODS: Concentrated stock mixtures, 10-fold dilutions of these mixtures in human serum albumin-saline diluent, and analogous single-extract controls were analyzed for major allergen concentrations (cat Fel d 1, dog dander Can f 1) and multiallergen IgE-binding potencies (dog epithelia, D farinae, cockroach mix) after storage for 3, 6, 9, and 12 months at 2°C to 8°C. RESULTS: The selected immunoassays were specific for individual target extracts in the 5-component mixtures and exhibited analytical sensitivities sufficient for evaluation of both the concentrated and diluted indoor allergen formulas. All control samples except diluted cockroach extract had near-complete stabilities during refrigerated storage. Mixtures that contained cat, dog epithelia, dog dander, and D farinae extracts exhibited favorable mixing compatibilities in 1:1 (vol/vol) concentrates (47.5% glycerin) and 1:10 (vol/vol) dilutions (4.75% glycerin), relative to corresponding control sample reactivities. Cockroach allergens in both 1:1 (vol/vol) and 1:10 (vol/vol) concentrations were stabilized significantly by mixing with the other 4 indoor allergen extracts. CONCLUSION: Extracts in mixtures that contained 5 common sources of indoor allergens possess favorable stabilities and mixing compatibilities and support the practice of combining these products in the same patient treatment formulations for subcutaneous immunotherapy.

Evolution of Growth Hormone Devices: Matching Devices with Patients.

Self-injection of growth hormone (GH) by children with GH deficiency can be problematic. They may have difficulty manipulating injection devices or preparing medication, and injections can be painful and create anxiety. Adherence to daily GH injections optimizes treatment benefit. Studies indicate that injection pens or needle-free devices enable easy self-injection by children, minimize medication reconstitution and storage requirements, and reduce injection pain. Newer GH delivery devices potentially encourage improved patient adherence. Reviewing features of GH devices will help nurses decide which GH device best fits the needs and abilities of pediatric patients. We searched recent medical literature about GH device development, about device-associated patient preferences and treatment adherence, and comparisons among GH devices. We concluded that improved awareness of the strengths and limitations of GH devices will enable nurses to guide families in selecting and using GH devices, improving adherence and outcomes, and helping children reach full growth potential.

Structure and Function of the Hypertension Variant A486V of G Protein-coupled Receptor Kinase 4.

G-protein-coupled receptor (GPCR) kinases (GRKs) bind to and phosphorylate GPCRs, initiating the process of GPCR desensitization and internalization. GRK4 is implicated in the regulation of blood pressure, and three GRK4 polymorphisms (R65L, A142V, and A486V) are associated with hypertension. Here, we describe the 2.6 Å structure of human GRK4α A486V crystallized in the presence of 5'-adenylyl ß,γ-imidodiphosphate. The structure of GRK4α is similar to other GRKs, although slight differences exist within the RGS homology (RH) bundle subdomain, substrate-binding site, and kinase C-tail. The RH bundle subdomain and kinase C-terminal lobe form a strikingly acidic surface, whereas the kinase N-terminal lobe and RH terminal subdomain surfaces are much more basic. In this respect, GRK4α is more similar to GRK2 than GRK6. A fully ordered kinase C-tail reveals interactions linking the C-tail with important determinants of kinase activity, including the αB helix, αD helix, and the P-loop. Autophosphorylation of wild-type GRK4α is required for full kinase activity, as indicated by a lag in phosphorylation of a peptide from the dopamine D1 receptor without ATP preincubation. In contrast, this lag is not observed in GRK4α A486V. Phosphopeptide mapping by mass spectrometry indicates an increased rate of autophosphorylation of a number of residues in GRK4α A486V relative to wild-type GRK4α, including Ser-485 in the kinase C-tail.

Synthesis and Evaluation of Heterocyclic Catechol Mimics as Inhibitors of Catechol-O-methyltransferase (COMT).

3-Hydroxy-4-pyridinones and 5-hydroxy-4-pyrimidinones were identified as inhibitors of catechol-O-methyltransferase (COMT) in a high-throughput screen. These heterocyclic catechol mimics exhibit potent inhibition of the enzyme and an improved toxicity profile versus the marketed nitrocatechol inhibitors tolcapone and entacapone. Optimization of the series was aided by X-ray cocrystal structures of the novel inhibitors in complex with COMT and cofactors SAM and Mg(2+). The crystal structures suggest a mechanism of inhibition for these heterocyclic inhibitors distinct from previously disclosed COMT inhibitors.

Improving resident handoffs for children transitioning from the intensive care unit.

BACKGROUND AND OBJECTIVE: Handoffs ensure patient safety during patient care transitions in the hospital setting. At our institution, verbal handoffs communicated by resident physicians are suggested practice for patients transferring from the PICU to the hospital medicine (HM) service. Despite their importance, these verbal handoffs occurred only 76% of the time before patient arrival on HM units. Our goal was to increase the completion rate of verbal handoffs to 100% within 5 months. METHODS: Baseline data were collected in a daily survey of HM residents. Interventions were developed and tested on small, incremental change cycles. Key interventions included education about the importance of handoffs, standardization of the handoff process, standardization of handoff documentation, and identification and mitigation of handoff documentation failures. We tracked handoff completion rates by using statistical control charts. After success with improving the completion rate of patient handoffs to the HM service, we applied our process to handoffs from the PICU to all inpatient services. RESULTS: Median completion of verbal patient handoff increased from 76% to 100% within 6 weeks, with improvement sustained for 15 months. Physician compliance with electronic medical record documentation increased from 58% to 94% within 8 months. After spreading to all patients transferring out of the PICU, documentation of patient handoffs increased from 76% to 94% in 5 months. CONCLUSIONS: A system using improvement science methods was successful in increasing the reliability of resident verbal patient handoffs. Consistent documentation and internal redundancy with checklists were associated with sustained improvement.

Mixing compatibilities of Aspergillus and American cockroach allergens with other high-protease fungal and insect extracts.

BACKGROUND: Recent studies have shown that Alternaria and German cockroach allergens can be degraded by endogenous proteases from other insect and fungal extracts when combined for immunotherapy, but data supporting the compatibilities of other high-protease products in comparable mixtures have not been reported. OBJECTIVE: To assess the stabilities and compatibilities of Aspergillus fumigatus and American cockroach allergens after mixing with protease-rich extracts from other insects or fungi at concentrations similar to those recommended for subcutaneous immunotherapy. METHODS: Mixtures containing A fumigatus, American cockroach, and other fungal or insect extracts were evaluated by quantitative (enzyme-linked immunosorbent assays) and qualitative (immunoblotting) methods. Test mixtures and control samples at 10% to 50% glycerin concentrations were analyzed after storage for up to 12 months at 2°C to 8°C. RESULTS: Moderate to high recoveries of Aspergillus extract activities were retained in control samples and extract mixtures under all conditions examined. American cockroach extract controls were partly degraded at 10% to 25% glycerin, and cockroach allergen compatibilities were decreased significantly in mixtures with several fungal extracts at 25% glycerin. Mixing with other insects did not compromise the stability of American cockroach allergens at 25% to 50% glycerin. CONCLUSION: Aspergillus extracts exhibited favorable stabilities after mixing with other high-protease products. American cockroach extract potencies were unstable in less than 50% glycerin, even in the absence of other protease-containing allergens, and were destabilized in mixtures with several fungal extracts. Addition of fungal and insect extracts to separate treatment vials or preparation of fungal-insect mixtures at elevated glycerin concentrations might be necessary to produce compatible patient formulations for allergen immunotherapy injections.

Plasticity and evolution of (+)-3-carene synthase and (-)-sabinene synthase functions of a sitka spruce monoterpene synthase gene family associated with weevil resistance.

The monoterpene (+)-3-carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3-carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3-carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multiproduct (+)-3-carene synthase-like genes of Sitka spruce include the three (+)-3-carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (-)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3-carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by (+)-3-carene in PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3, or (-)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3-carene, whereas phenylalanine promotes (-)-sabinene. Homology modeling predicts that position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against insects.

Transcriptome resources and functional characterization of monoterpene synthases for two host species of the mountain pine beetle, lodgepole pine (Pinus contorta) and jack pine (Pinus banksiana).

BACKGROUND: The mountain pine beetle (MPB, Dendroctonus ponderosae) epidemic has affected lodgepole pine (Pinus contorta) across an area of more than 18 million hectares of pine forests in western Canada, and is a threat to the boreal jack pine (Pinus banksiana) forest. Defence of pines against MPB and associated fungal pathogens, as well as other pests, involves oleoresin monoterpenes, which are biosynthesized by families of terpene synthases (TPSs). Volatile monoterpenes also serve as host recognition cues for MPB and as precursors for MPB pheromones. The genes responsible for terpene biosynthesis in jack pine and lodgepole pine were previously unknown. RESULTS: We report the generation and quality assessment of assembled transcriptome resources for lodgepole pine and jack pine using Sanger, Roche 454, and Illumina sequencing technologies. Assemblies revealed transcripts for approximately 20,000 - 30,000 genes from each species and assembly analyses led to the identification of candidate full-length prenyl transferase, TPS, and P450 genes of oleoresin biosynthesis. We cloned and functionally characterized, via expression of recombinant proteins in E. coli, nine different jack pine and eight different lodgepole pine mono-TPSs. The newly identified lodgepole pine and jack pine mono-TPSs include (+)-α-pinene synthases, (-)-α-pinene synthases, (-)-ß-pinene synthases, (+)-3-carene synthases, and (-)-ß-phellandrene synthases from each of the two species. CONCLUSION: In the absence of genome sequences, transcriptome assemblies are important for defence gene discovery in lodgepole pine and jack pine, as demonstrated here for the terpenoid pathway genes. The product profiles of the functionally annotated mono-TPSs described here can account for the major monoterpene metabolites identified in lodgepole pine and jack pine.

Evolution of conifer diterpene synthases: diterpene resin acid biosynthesis in lodgepole pine and jack pine involves monofunctional and bifunctional diterpene synthases.

Diterpene resin acids (DRAs) are major components of pine (Pinus spp.) oleoresin. They play critical roles in conifer defense against insects and pathogens and as a renewable resource for industrial bioproducts. The core structures of DRAs are formed in secondary (i.e. specialized) metabolism via cycloisomerization of geranylgeranyl diphosphate (GGPP) by diterpene synthases (diTPSs). Previously described gymnosperm diTPSs of DRA biosynthesis are bifunctional enzymes that catalyze the initial bicyclization of GGPP followed by rearrangement of a (+)-copalyl diphosphate intermediate at two discrete class II and class I active sites. In contrast, similar diterpenes of gibberellin primary (i.e. general) metabolism are produced by the consecutive activity of two monofunctional class II and class I diTPSs. Using high-throughput transcriptome sequencing, we discovered 11 diTPS from jack pine (Pinus banksiana) and lodgepole pine (Pinus contorta). Three of these were orthologous to known conifer bifunctional levopimaradiene/abietadiene synthases. Surprisingly, two sets of orthologous PbdiTPSs and PcdiTPSs were monofunctional class I enzymes that lacked functional class II active sites and converted (+)-copalyl diphosphate, but not GGPP, into isopimaradiene and pimaradiene as major products. Diterpene profiles and transcriptome sequences of lodgepole pine and jack pine are consistent with roles for these diTPSs in DRA biosynthesis. The monofunctional class I diTPSs of DRA biosynthesis form a new clade within the gymnosperm-specific TPS-d3 subfamily that evolved from bifunctional diTPS rather than monofunctional enzymes (TPS-c and TPS-e) of gibberellin metabolism. Homology modeling suggested alterations in the class I active site that may have contributed to their functional specialization relative to other conifer diTPSs.

Identification of genes in Thuja plicata foliar terpenoid defenses.

Thuja plicata (western redcedar) is a long-lived conifer species whose foliage is rarely affected by disease or insect pests, but can be severely damaged by ungulate browsing. Deterrence to browsing correlates with high foliar levels of terpenoids, in particular the monoterpenoid α-thujone. Here, we set out to identify genes whose products may be involved in the production of α-thujone and other terpenoids in this species. First, we generated a foliar transcriptome database from which to draw candidate genes. Second, we mapped the storage of thujones and other terpenoids to foliar glands. Third, we used global expression profiling to identify more than 600 genes that are expressed at high levels in foliage with glands, but can either not be detected or are expressed at low levels in a natural variant lacking foliar glands. Fourth, we used in situ RNA hybridization to map the expression of a putative monoterpene synthase to the epithelium of glands and used enzyme assays with recombinant protein of the same gene to show that it produces sabinene, the monoterpene precursor of α-thujone. Finally, we identified candidate genes with predicted enzymatic functions for the conversion of sabinene to α-thujone. Taken together, this approach generated both general resources and detailed functional characterization in the identification of genes of foliar terpenoid biosynthesis in T. plicata.

Improving situation awareness to reduce unrecognized clinical deterioration and serious safety events.

BACKGROUND AND OBJECTIVE: Failure to recognize and treat clinical deterioration remains a source of serious preventable harm for hospitalized patients. We designed a system to identify, mitigate, and escalate patient risk by using principles of high-reliability organizations. We hypothesized that our novel care system would decrease transfers determined to be unrecognized situation awareness failures events (UNSAFE). These were defined as any transfer from an acute care floor to an ICU where the patient received intubation, inotropes, or ≥ 3 fluid boluses in first hour after arrival or before transfer. METHODS: The setting for our observational time series study was a quaternary care children's hospital. Before initiating tests of change, 2 investigators reviewed recent serious safety events (SSEs) and floor-to-ICU transfers. Collectively, 5 risk factors were associated with each event: family concerns, high-risk therapies, presence of an elevated early warning score, watcher/clinician gut feeling, and communication concerns. Using the model for improvement, an intervention was developed and tested to reliably and proactively identify patient risk and mitigate that risk through unit-based huddles. A 3-times daily inpatient huddle was added to ensure risks were escalated and addressed. Later, a "robust" and explicit plan for at-risk patients was developed and spread. RESULTS: The rate of UNSAFE transfers per 10,000 non-ICU inpatient days was significantly reduced from 4.4 to 2.4 over the study period. The days between inpatient SSEs also increased significantly. CONCLUSIONS: A reliable system to identify, mitigate, and escalate risk was associated with a near 50% reduction in UNSAFE transfers and SSEs.

Characterization of non-nitrocatechol pan and isoform specific catechol-O-methyltransferase inhibitors and substrates.

Reduced dopamine neurotransmission in the prefrontal cortex has been implicated as causal for the negative symptoms and cognitive deficit associated with schizophrenia; thus, a compound which selectively enhances dopamine neurotransmission in the prefrontal cortex may have therapeutic potential. Inhibition of catechol-O-methyltransferase (COMT, EC 2.1.1.6) offers a unique advantage, since this enzyme is the primary mechanism for the elimination of dopamine in cortical areas. Since membrane bound COMT (MB-COMT) is the predominant isoform in human brain, a high throughput screen (HTS) to identify novel MB-COMT specific inhibitors was completed. Subsequent optimization led to the identification of novel, non-nitrocatechol COMT inhibitors, some of which interact specifically with MB-COMT. Compounds were characterized for in vitro efficacy versus human and rat MB and soluble (S)-COMT. Select compounds were administered to male Wistar rats, and ex vivo COMT activity, compound levels in plasma and cerebrospinal fluid (CSF), and CSF dopamine metabolite levels were determined as measures of preclinical efficacy. Finally, novel non-nitrocatechol COMT inhibitors displayed less potent uncoupling of the mitochondrial membrane potential (MMP) compared to tolcapone as well as nonhepatotoxic entacapone, thus mitigating the risk of hepatotoxicity.

Molecular cloning and biochemical characterization of the UDP-glucose: flavonoid 3-O-glucosyltransferase from Concord grape (Vitis labrusca).

Glucosylation of anthocyanidin substrates at the 3-O-position is crucial for the red pigmentation of grape berries and wine. The gene that encodes the enzyme involved in this reaction has been cloned from Vitis labrusca cv. Concord, heterologously expressed, and the recombinant enzyme (rVL3GT) was characterized. VL3GT has 96% amino acid sequence identity with Vitis vinifera VV3GT and groups phylogenetically with several other flavonoid 3-O-glycosyltransferases. In vitro substrate specificity studies and kinetic analyses of rVL3GT indicate that this enzyme preferentially glucosylates cyanidin as compared with quercetin. Crude protein extracts from several Concord grape tissues were assayed for glucosyltransferase activity with cyanidin and quercetin as acceptor substrates. A comparison of the VL3GT activities toward with these substrates showed that the 3GT enzyme activity is consistent with the expression of VL3GT in these tissues and is coincident with the biosynthesis of anthocyanins in both location and developmental stages. Enzyme activities in grape mesocarp, pre-veraison exocarp, leaf, flower bud, and flower tissues glucosylated quercetin but not cyanidin at high rates, suggesting the presence of additional enzymes which are able to glucosylate the 3-O-position of flavonols with higher specificity than anthocyanidins.

Molecular cloning and biochemical characterization of three Concord grape (Vitis labrusca) flavonol 7-O-glucosyltransferases.

Grapes berries produce and accumulate many reactive secondary metabolites, and encounter a wide range of pathogen- and human-derived xenobiotic compounds. The enzymatic glucosylation of these metabolites changes their reactivity, stability and subcellular location. Two ESTs with more than 90% nucleotide sequence identity to three full-length glucosyltransferases are expressed in several grape tissues. The full-length clones have more than 60% amino acid sequence similarity to previously characterized flavonoid 7-O-glucosyltransferases, catechin O-glucosyltransferases and anthocyanin 5-O-glucosyltransferases. In vitro, these enzymes glucosylate flavonols and the xenobiotic 2,4,5-trichlorophenol (TCP). Kinetic analysis indicates that TCP is the preferred substrate for these enzymes, while expression analysis reveals variable transcription of these genes in grape leaves, flowers and berry tissues. The in vivo role of these Vitis labrusca glucosyltransferases is discussed.

An integrated genomic, proteomic and biochemical analysis of (+)-3-carene biosynthesis in Sitka spruce (Picea sitchensis) genotypes that are resistant or susceptible to white pine weevil.

Conifers are extremely long-lived plants that have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. In these species, terpenoid diversity is determined by the size and composition of the terpene synthase (TPS) gene family and the single- and multi-product profiles of these enzymes. The monoterpene (+)-3-carene is associated with resistance of Sitka spruce (Picea sitchensis) to white pine weevil (Pissodes strobi). We used a combined genomic, proteomic and biochemical approach to analyze the (+)-3-carene phenotype in two contrasting Sitka spruce genotypes. Resistant trees produced significantly higher levels of (+)-3-carene than susceptible trees, in which only trace amounts were detected. Biosynthesis of (+)-3-carene is controlled, at the genome level, by a small family of closely related (+)-3-carene synthase (PsTPS-3car) genes (82-95% amino acid sequence identity). Transcript profiling identified one PsTPS-3car gene (PsTPS-3car1) that is expressed in both genotypes, one gene (PsTPS-3car2) that is expressed only in resistant trees, and one gene (PsTPS-3car3) that is expressed only in susceptible trees. The PsTPS-3car2 gene was not detected in genomic DNA of susceptible trees. Target-specific selected reaction monitoring confirmed this pattern of differential expression of members of the PsTPS-3car family at the proteome level. Kinetic characterization of the recombinant PsTPS-3car enzymes identified differences in the activities of PsTPS-3car2 and PsTPS-3car3 as a factor contributing to the different (+)-3-carene profiles of resistant and susceptible trees. In conclusion, variation of the (+)-3-carene phenotype is controlled by copy number variation of PsTPS-3car genes, variation of gene and protein expression, and variation in catalytic efficiencies.