| Rare ginsenosides are a group of dammarane tetracyclic triterpenoid sapanins and exhibit anticancer,antitumor,and immune-system-enhancing activities.Glycosylation mediated by uridine diphosphate-dependent glycosyltransferases(UGT)is the last and key rate-limiting reaction in the bosynthesis of rare ginsenosides.Bs-YjiC from Bacillus subtilis is a robust and versatile UGT that can glycosylate both the free C3-OH of PPD to synthesize ginsenoside Rh2 and unnatural ginsenoside F12.To understand the molecular mechanism underlying the substrate promiscuity and unique regiospecificity of Bs-YjiC,the crystal structure of Bs-YjiC and its complex with UDP were solved.Moreover,the molecular mechanism for the interactions between Bs-YjiC and PPD and ginsenoside Rh2 was determined by molecular docking simulations.The Bs-YjiC gene was amplified and cloned into the p ET28 a,then transformed into E.coli BL21(DE3).The recombinant E.coli strains were cultured at 16℃ for 16 h in the presence of 0.3 m M IPTG.Bs-YjiC was purified by by an AKTA Purifier FPLC system(GE Healthcare)coupled with a Ni NTA column and then subjected to a DEAE column and eluted with a gradient of 0-500 m M Na Cl.The 46 k Da Bs-YjiC was purified to apparent homogeneity and was found to exist as a monomer in solution,as confirmed by SDS-PAGE and static light scattering.High-quality Bs-YjiC crystals were obtained through crystal culture and crystal conditions optimization.The optimized crystal growth conditions were as follows: 1.3 M trisodium citrate,0.1 M Bis-Tris propane,pH 7.0.The structure of Bs-YjiC and Bs YjiC/UDP binary complex was analyzed by X-ray crystal diffraction technique with the resolution of2.18 (?) and 2.44 (?),respectively.The overall structure of Bs-YjiC is a classic GT-B folded configuration,which is composed of two significantly different Rossmann domains connected by a flexible peptide chain.The sugar receptor binding region is located in the hydrophobic pocket of the N-terminal domain of UGT enzyme,while the glycosyl donor binding region is located in a deep pocket of the C-terminal domain.The N-terminal domain consists of 6 αhelices and 7 parallel β chains surrounded by α helices.The C-terminal domain consists of six parallel β chains flanked by eight α helices.The α7 and α8 helices on the C-terminal cross the junction of the two domains and extend to the N-terminal domain.The distance between H16 and D106 is 3.0 (?),which is beneficial to increase the ability of the imidazole ring of H16 to accept the sugar accepter proton,and the proximity of H16 to the C1 of the UDP-glucose donor(4.3 (?))is conducive to the deprotonated sugar donor to attack the C1 site of UDP-glucose.The molecular model of Bs-YjiC/UDPG binary complex with PPD and Rh2 was analyzed by Libdock modeling software.It was found that the wide sugar receptor binding pocket of Bs-YjiC might determine the unique region selection specificity of Bs-YjiC for PPD and Rh2.The distance between the catalytic sites of H16 and C3-OH of PPD and C12-OH of Rh2 are 2.7 (?) and 3.2 (?),respectively in the complex structure,which contribute to the deprotonation and glycosylation of C3-OH of PPD and C12-OH of Rh2 by H16.While the distance between the C20-OH of Rh2 and the catalytic site of H16 is 4.7 (?),which is not conducive to H16 attacking the C20-OH of Rh2.The above structural simulation analysis is highly consistent with the data verified by previous biochemical experiments that Bs-YjiC can only catalyze the C3-OH and C12-OH of PPD and Rh2,but cannot catalyze the glycosylation of C20-OH.In the present study,the structures of glycosyltransferase Bs-YjiC from Bacillus subtilis and its complex with UDP were resolved.Moreover,the reaction mechanism of Bs-YjiC for its unique regiospecificity toward PPD was determined by molecular model analysis for its large sugar receptor binding pocket.These results facilitate the rational design and molecular modification of Bs-YjiC,and biosynthesis of natural bioactive products. |
PDF Full Text Request