Flavonoids are ubiquitous secondary plant metabolites which function as protectants against UV light and pathogens and are involved in the attraction of pollinators as well as seed and fruit dispersers. The hydroxylation pattern of the B-ring of flavonoids is determined by the activity of two members of the vast and versatile cytochrome P450 protein (P450) family, the flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H). Phylogenetic analysis of known sequences of F3'H and F3'5'H indicated that F3'5'H was recruited from F3'H before the divergence of angiosperms and gymnosperms. Seven cDNAs were isolated from species of the Asteraceae family, all of which were predicted to code for F3'Hs based on their sequences. The recombinant proteins of four of the heterologously in yeast expressed cDNAs exhibited the expected F3'H activity but surprisingly, three recombinant proteins showed F3'5'H activity. Phylogenetic analyses indicated the independent evolution of an Asteraceae-specific F3'5'H. Furthermore, sequence analysis of these unusual F3'5'H cDNAs revealed an elevated rate of nonsynonymous substitutions as typically found for duplicated genes acquiring new functions. Since F3'5'H is necessary for the synthesis of 3',4',5'-hydroxylated delphinidin-derivatives, which normally provide the basis for purple to blue flower colours, the evolution of an Asteraceae-specific F3'5'H probably reflects the adaptive value of efficient attraction of insect pollinators.
Flavonoid 30 -hydroxylase (F3 0 H) and flavonoid 3 0 ,5 0 -hydroxylase (F3 0 5 0 H) are cytochrome P450 enzymes and determine the B-ring hydroxylation pattern of flavonoids by introducing hydroxyl groups at the 3 0 -or the 3 0 -and 5 0 -position, respectively. Sequence identity between F3 0 H and F3 0 5 0 H is generally low since their divergence took place early in the evolution of higher plants. However, in the Asteraceae the family-specific evolution of an F3 0 5 0 H from an F3 0 H precursor occurred, and consequently sequence identity is substantially higher. We used this phenomenon for alignment studies, in order to identify regions which could be involved in determining substrate specificity and functionality. Subsequent construction and expression of chimeric genes indicated that substrate specificity of F3 0 H and F3 0 5 0 H is determined near the N-terminal end and the functional difference between these two enzymes near the C-terminal end. The impact on function of individual amino acids located in substrate recognition site 6 (SRS6) was further tested by site-directed mutagenesis. Most interestingly, a conservative Thr to Ser exchange at position 487 conferred additional 5 0 -hydroxylation activity to recombinant Gerbera hybrida F3 0 H, whereas the reverse substitution transformed recombinant Osteospermum hybrida F3 0 5 0 H into an F3 0 H with low remaining 5 0 -hydroxylation activity. Since the physicochemical properties of Thr and Ser are highly similar, the difference in size appears to be the main factor contributing to functional difference. The results further suggest that relatively few amino acids exchanges were required for the evolutionary extension of 3 0 -to 3 0 ,5 0 -hydroxylation activity.
Objective: Neurokinin (NK)-3 and NK-1 receptors have been implicated in the etiology of vasomotor symptoms (VMS) and sleep disturbances associated with menopause. This phase 2b, adaptive, dose-range finding study aimed to assess the efficacy and safety of multiple doses of elinzanetant (NT-814), a selective NK-1,3 receptor antagonist, in women experiencing VMS associated with menopause, and investigate the impact of elinzanetant on sleep and quality of life.Methods: Postmenopausal women aged 40 to 65 years who experienced seven or more moderate-to-severe VMS per day were randomized to receive elinzanetant 40, 80, 120, or 160 mg or placebo once daily using an adaptive design algorithm. Coprimary endpoints were reduction in mean frequency and severity of moderate-to-severe VMS at weeks 4 and 12. Secondary endpoints included patient-reported assessments of sleep and quality of life.Results: Elinzanetant 120 mg and 160 mg achieved reductions in VMS frequency versus placebo from week 1 throughout 12 weeks of treatment. Least square mean reductions were statistically significant versus placebo at both primary endpoint time points for elinzanetant 120 mg (week 4: −3.93 [SE, 1.02], P < 0.001; week 12: −2.95 [1.15], P = 0.01) and at week 4 for elinzanetant 160 mg (−2.63 [1.03]; P = 0.01). Both doses also led to clinically meaningful improvements in measures of sleep and quality of life. All doses of elinzanetant were well tolerated.Conclusions: Elinzanetant is an effective and well-tolerated nonhormone treatment option for postmenopausal women with VMS and associated sleep disturbance. Elinzanetant also improves quality of life in women with VMS.
Multiple F3'5'H evolution from F3'H has occurred in dicotyledonous plants. Efficient pollinator attraction is probably the driving force behind, as this allowed for the synthesis of delphinidin-based blue anthocyanins. The cytochrome P450-dependent monooxygenases flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) hydroxylate the B-ring of flavonoids at the 3'- and 3'- and 5'-position, respectively. Their divergence took place early in plant evolution. While F3'H is ubiquitously present in higher plants, the distribution of F3'5'H is scattered. Here, we report that F3'5'H has repeatedly evolved from F3'H precursors at least four times in dicotyledonous plants: In the Asteraceae, we identified F3'5'Hs specific for the subfamilies Cichorioideae and Asteroideae, and additionally an F3'5'H that seems to be specific for the genus Echinops of the subfamily Carduoideae; moreover, characterisation of a sequence from Billardiera heterophylla (formerly Sollya heterophylla) (Pittosporaceae) showed that the independent evolution of an F3'5'H has occurred at least once also in another family. The evolution of F3'5'H from an F3'H precursor represents a gain of enzymatic function, probably triggered by an amino acid change at one position of substrate recognition site 6. The gain of F3'5'H activity allows for the synthesis of delphinidin-based anthocyanins which usually provide the basis for lilac to blue flower colours. Therefore, the need for an efficient pollinator attraction is probably the driving force behind the multiple F3'5'H evolution.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.