The Catalytic Mechanism Of Sulfoglycosidase Bt4394 And Its Inhibitor Activity Evaluation

The mucin secreted by the intestine contains highly sulfated oligosaccharide side chains.This highly sulfated mucin has the stronger lubricating and barrier effect than ordinary mucin,and may prevent excessive degradation of mucin by intestinal flora.However,some bacteria have a mucin desulfurization effect and could remove the sulfated glycans from mucin.Some studies have also found that some bacterial mucus desulfurase levels in the stool of patients with ulcerative colitis are raised.At present,the structure,function and substrate binding mode of Bbh II from Bifidobacterium,SGL from Prevotella strain RS2 and Bt4394 from Bacteroides are not well understood.Therefore,studying the catalytic mechanism of sulfoglycosidase expressed by the microbial flora in the intestinal tract is of great significance for understanding the relationship between microorganisms,mucus layer and host.The possible catalytic mechanism of glycosidase is generally divided into three types,namely,the single displacement mechanism of the inverting glycosidase,the double displacement mechanism of retaining glycosidase and the substrate-assisted catalytic mechanism.Bt4394 from Bacteroides belongs to the GH20 family of glucoside hydrolase.The catalytic mechanism of glycosidases in this family is the substrate-assisted catalytic mechanism involving the synthesis and decomposition of oxazoline rings.Herein,this thesis studies the catalytic mechanism of Bt4394 from Bacteroides and a preliminary evaluation of the activity of its inhibitors was carried out,which contribute to the screening and development of new Chinese medicine effective substances to protect the mucus layer.The specific research contents are as follows:（1）Using coumarin derivatives 4-methylumbelliferone as the fluorophore,we designed and synthesized N-acetylaminoglycoside substrates with different fluorine substitutions at the2-position of glucosamine.The growth rate of the fluorescence intensity generated by the hydrolysis of the substrate within 60 s was used to determine the reactivity of Bt4394 to achieve visual effects.The kinetic experiment results show that as the number of substitutions of F atoms increases,the hydrolysis rate of glycosidases is significantly reduced.Because F is a strong electron-withdrawing group,the greater the number of substitutions,the stronger the electron-withdrawing effect.That affects the attack of carbonyl oxygen on the anomeric carbon and slows down the synthesis rate of the intermediate oxazoline ring.Above experimental results show that the hydrolysis mechanism of Bt4394 is the substrate-assisted catalytic mechanism.（2）Based on the catalytic mechanism of Bt4394 and the known inhibitor NAG-thiazoline of the GH20 family,we designed and synthesized 6S-NAG-thiazoline as glycosidase inhibitors.Finally,the IC₅₀ of 6S-NAG-thiazoline for Bt4394 was detected by fluorescence intensity,it was found that 6S-NAG-thiazoline could bind to Bt4394competitively(IC₅₀=4.8μM);subsequently,the known inhibitor NAG-thiazoline of the GH20family was used as the reference compound,the affinity K_i values of 6S-NAG-thiazoline and NAG-thiazoline to Bt4394 were determined to be 4.0μM and 154.7μM.The two inhibitors with similar structures have obvious differences in affinity,which indicating that the6-position sulfonic acid group is the specific recognition groups of Bt4394.Based on the above experimental results,6S-NAG-thiazoline can also be used as a model drug for Chinese medicine screening inhibitor of Bt4394.