YfiBNR is a recently identified bis-（3＇-5＇）-cyclic dimeric GMP （c-di-GMP） signaling system in opportunistic pathogens. It is a key regulator of biofilm formation, which is correlated with prolonged persistence o...YfiBNR is a recently identified bis-（3＇-5＇）-cyclic dimeric GMP （c-di-GMP） signaling system in opportunistic pathogens. It is a key regulator of biofilm formation, which is correlated with prolonged persistence of infection and antibiotic drug resistance. In response to cell stress, YfiB in the outer membrane can sequester the pariplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. However, the detailed regulatory mechanism remains elusive. Here, we report the crystal struc- tures of YfiB alone and of an active mutant YfiB^L43P complexed with YfiR with 2：2 stoichiometry. Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiB^L43P adopts a stretched conformation allowing activated YfiB to penebate the peptidoglycan （PG） layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YflB, suggesting a tight correlation between PG binding and YfiB activation. In addition, our crystallographic analyses revealed that YflR binds Vitamin B6 （VB6） or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system.展开更多
By employing TCLs (thin cell layers) culture, the floral gradient in flowering tobacco of different developmental stages was confirmed. The TCLs from early flowering tobacco regenerated more floral buds than those fro...By employing TCLs (thin cell layers) culture, the floral gradient in flowering tobacco of different developmental stages was confirmed. The TCLs from early flowering tobacco regenerated more floral buds than those from the tobacco plants in full blooming or fruiting stages. Analysis of free amino acid levels revealed the acropetal gradient of Pro in flowering tobacco stem. L-Pro. L-Trp. D,L-Met and L-Arg were respectively added into the culture medium for testing their influence on floral bud formation from tobacco pedicel segments. Only L-Trp evidently enhanced the floral bud neoformation.展开更多
It has previously been shown that tryptophan, the biosynthesis precursor ofPeganum harmala alkaloids, and its derivatives have anti-TMV activity bothin vitro and in vivo. Further exploration of this led to the identif...It has previously been shown that tryptophan, the biosynthesis precursor ofPeganum harmala alkaloids, and its derivatives have anti-TMV activity bothin vitro and in vivo. Further exploration of this led to the identification of NK0238as a highly effective agent for the prevention and control of diseases caused byplant viruses, but the existing routes are unsuitable for its large-scale synthesis.This study optimized a route for two-step synthesis of this virucide candidate viareaction of L-tryptophan with triphosgene to produce L-tryptophan-N-carboxylicanhydride, which then reacts with n-octylamine to give NK0238 at up to 94%yield and nearly 97% HPLC purity. In addition, the route was used for thepreparation of NK0238 on a > 40 g scale permitting further assessment of itsantivirus activity in the greenhouse and field experiments, and toxicity tests.NK0238 exhibited useful antiviral activities against a variety of viruses both ingreenhouse and field experiments. The toxicity tests showed that NK0238 wasnot acutely toxic to birds, fish, honey bees and silkworms. The optimized routeprovides a solid foundation for its large-scale synthesis and subsequent efficacyand toxicity studies, its excellent activity and safety make NK0238 a promisingdrug candidate for further development.展开更多
文摘YfiBNR is a recently identified bis-（3＇-5＇）-cyclic dimeric GMP （c-di-GMP） signaling system in opportunistic pathogens. It is a key regulator of biofilm formation, which is correlated with prolonged persistence of infection and antibiotic drug resistance. In response to cell stress, YfiB in the outer membrane can sequester the pariplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. However, the detailed regulatory mechanism remains elusive. Here, we report the crystal struc- tures of YfiB alone and of an active mutant YfiB^L43P complexed with YfiR with 2：2 stoichiometry. Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiB^L43P adopts a stretched conformation allowing activated YfiB to penebate the peptidoglycan （PG） layer and access YfiR. YfiBL43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YflB, suggesting a tight correlation between PG binding and YfiB activation. In addition, our crystallographic analyses revealed that YflR binds Vitamin B6 （VB6） or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiBL43P-induced biofilm formation. Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system.
文摘By employing TCLs (thin cell layers) culture, the floral gradient in flowering tobacco of different developmental stages was confirmed. The TCLs from early flowering tobacco regenerated more floral buds than those from the tobacco plants in full blooming or fruiting stages. Analysis of free amino acid levels revealed the acropetal gradient of Pro in flowering tobacco stem. L-Pro. L-Trp. D,L-Met and L-Arg were respectively added into the culture medium for testing their influence on floral bud formation from tobacco pedicel segments. Only L-Trp evidently enhanced the floral bud neoformation.
基金The authors are grateful to the National Natural Science Foundation of China(21977056,21732002)the Fundamental Research Funds for the Central Universities,Nankai University(63201043)for generous financial support for their programs.
文摘It has previously been shown that tryptophan, the biosynthesis precursor ofPeganum harmala alkaloids, and its derivatives have anti-TMV activity bothin vitro and in vivo. Further exploration of this led to the identification of NK0238as a highly effective agent for the prevention and control of diseases caused byplant viruses, but the existing routes are unsuitable for its large-scale synthesis.This study optimized a route for two-step synthesis of this virucide candidate viareaction of L-tryptophan with triphosgene to produce L-tryptophan-N-carboxylicanhydride, which then reacts with n-octylamine to give NK0238 at up to 94%yield and nearly 97% HPLC purity. In addition, the route was used for thepreparation of NK0238 on a > 40 g scale permitting further assessment of itsantivirus activity in the greenhouse and field experiments, and toxicity tests.NK0238 exhibited useful antiviral activities against a variety of viruses both ingreenhouse and field experiments. The toxicity tests showed that NK0238 wasnot acutely toxic to birds, fish, honey bees and silkworms. The optimized routeprovides a solid foundation for its large-scale synthesis and subsequent efficacyand toxicity studies, its excellent activity and safety make NK0238 a promisingdrug candidate for further development.
