Glycosyltransferases (GTs) catalyze the transfer of sugar moieties from activated donors to acceptor molecules, which are widely-used biocatalysts in the industrial biosynthesis of natural products. Members of the GT1 family exhibit remarkable substrate promiscuity, enabling glycosylation of diverse natural products; however, the underlying molecular mechanisms remain poorly understood. Here we integrated all-atom molecular dynamics simulations, bioinformatics and experimental mutations to elucidate the structural and dynamics mechanisms governing the substrate promiscuity of GT1 enzymes. Using the Bs YjiC from Bacillus subtilis as a representative, we interestingly found that its acceptor-binding pocket harbored two flexible binding subsites with distinct geometric features, which enabled structurally diverse substrates to be specifically recognized in different subsites. The site-directed mutagenesis at the subsites and in vitro glycosylation assays further demonstrated the critical role of subsite-mediated substrate recognition. Importantly, the existence of two subsites in the acceptor-binding pocket was found to be a general feature across nine different members of GT1 family. Overall, the characteristic features of the acceptor-binding pocket represent a conserved trait across the GT1 family and provide potential guidance for engineering GTs with tailored substrate specificities.
Xiaoli Dai,Yuanyuan He,Xinghui Han,Wen Sun,Jian Yu,Yating Lin,Yonghong Wang
Background: The insulin-like growth factor (IGF) system plays a vital role in regulating gonadotropin-releasing hormone (GnRH), whether the IGF2 can act on the GnRH neurons during the pubertal period is unclear.
Methods: Central precocious puberty (CPP) rats were induced by danazol, and when the rats met the first diestrus, they were euthanized and tissues were collected. GT1-7 cells were cultured and treated with 0, 1, 10 ng/mL IGF2 for 4 hours and the changes in GnRH were measured. Mice were injected intracerebroventricularly with IGF2 (15 ng/g, 5 μL) or with the same dose of phosphate buffered saline (PBS), after eight hours, they were euthanized and tissues collected.
Results: CPP rats had increased expression of IGF2 and GnRH mRNA and their respective proteins in the preoptic area (POA) of the hypothalamus. Treatment of GT1-7 cells with 10 ng/mL of IGF2 increased GnRH mRNA and protein expression, and GnRH concentration in the culture medium. Injection of IGF2 protein into the lateral ventricle of mice increased the expression of GnRH mRNA and protein in the POA. Conclusions: IGF2 may upregulate the synthesis of GnRH during the pubertal period, and may also take part in the pathology of CPP.
Yuan Yuan,Jian Yang,Xiaodan Yu,Luqi Huang,Shufang Lin
Recently, a new kind of Lonicera japonica (FLJ) tea well known for its red color, flavor, and taste has become popular in the market. This tea comes from the buds of Lonicera japonica Thunb. var. chinensis (Wats.) Bak. (rFLJ), a wild mutant of the Lonicerae plant. The present study aimed to identify and quantify, for the first time and in detail, the anthocyanin compounds in the buds of rFLJ. Anthocyanins were extracted with methanol–water–formic acid–trifluoroacetic acid (70:27:2:1, v/v/v/v) and quantified as 108.06 ± 4.32 mg/100 g of fresh weight (cyanidin-3-glucoside equivalent) by using the pH-differential method. Eight different anthocyanins were identified by HPLC–DAD–ESI-MS/MS analysis and were quantitated by HPLC–DAD analysis. The main anthocyanins in the buds of rFLJ were two non-acylated anthocyanins: cyanidin-3,5-diglucoside and cyanidin-3-glucoside. Moreover, orthologous genes in the biosynthesis of anthocyanins were cloned and their structure and transcript levels in the buds of FLJ and rFLJ were further compared. The results revealed a 12-residue insertion (IKPTIEGVLGII) in LjcDFR compared with LjDFR. Furthermore, transcript levels of DFR, LDOX, BZ1 and GT1 were decreased in the buds of FLJ compared with rFLJ, as constitute with the content of cyanidin-3-glucoside. Here, a rapid and effective method to identify and quantify all major anthocyanins in the buds of rFLJ is established. Furthermore, the results of this study may explain which anthocyanin produces the red color of rFJL, an important characteristic that distinguishes it from FLJ.
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