Rational Design To Improve The Activity Of CMP-sialic Acid Synthase Nmcss On Fluorescent Sialic Acid Substrates

Sialic acid?SA?is a general term for a family of negatively charged nine-carbon monosaccharides,which are widely distributed in vertebrates,plants and microorganisms.In organisms,sialic acid is usually expressed on the surface of cells and plays multiple functions in the regulation of the organism's immune system,viral infection as well as tumor development.Sialic acid metabolism in organisms is generally closely related to the two enzymes,CMP-sialic acid synthase?CSS?and sialyltransferase?ST?.Due to the important biological role of sialic acid,it has become an attractive research direction to label and image sialic acid on the cell surface of organisms in recent years.Metabolic glycan labeling based on bioorthogonal chemical reactions and chemical enzymatic labeling are two commonly used methods for cell glycan labeling.Both methods are initially labeled in two steps.The first step of metabolic glycan labeling is to express sialic acid analogues with bioorthogonal functional groups on the cell surface through the intracellular glycan metabolic system,and the second step is covalent labeling using complementary bioorthogonal probes.However,this technique has obvious limitations,first,it will cause uncontrollable large-scale disturbances to organisms,and second,the two-step method based on bioorthogonal reactions is relatively inefficient.Chemo-enzymatic labeling is performed by transferring CMP-sialic acid analogues with orthogonal reaction handles to the cell surface by sialyltransferases with a wide range of substrate recognition,followed by labeling by bioorthogonal reactions.Inevitably,it also has the problem of low labeling efficiency of bioorthogonal reactions.Later,one-step chemical microplate labeling techniques were developed to transfer CMP-sialic acid analogues of fluorophores directly to the cell surface for labeling by sialyltransferases.It greatly improves the efficiency of sialic acid labeling on the cell surface.However,because of the weak catalytic activity of CMP-sialic acid synthase?CSS?for fluorescent sialic acid analogues,fluorescent CMP-sialic acid analogue raw materials currently used for one-step chemical enzymatic labeling are mainly synthesized chemically.CMP-sialic acid analogues were synthesized using CMP-sialic acid synthase?CSS?followed by chemical synthesis of conjugated fluorophores.In this process,because of the poor stability of the CMP-sialic acid analog,it is very easy to hydrolyze,resulting in a low yield of generated fluorescent CMP-sialic acid analogues.Therefore,the use of protein modification technology to improve the catalytic activity of CMP-sialic acid synthase?CSS?for fluorescent sialic acid analogues is of great significance for the synthesis of a large number of fluorescent CMP-sialic acid analogue raw materials to improve the efficiency of one-step chemical enzymatic labeling.In this study,site-directed mutagenesis of existing N.meningitidis CMP-sialic acid synthase?Nm CSS?in the laboratory was performed based on a computer rational design to improve its enzymatic activity against the fluorescent sialic acid analogue ^FITC-SA.The main research contents are as follows:?1?^FITC-SA,a fluorescent sialic acid analogue,was chemically synthesized from9-NH2-SA and FITC.?2?^FITC-SA was molecularly docked to the Nm CSS crystal structure to obtain the preliminary binding mode of ^FITC-SA to Nm CSS.Analysis of binding patterns revealed that histidine H at position 138 and arginine R at position 173 had a potential hindering effect on Nm CSS binding to ^FITC-SA.?3?Site-directed mutagenesis was designed for histidine at position 138 and arginine at position 173 of Nm CSS to construct the mutant vectors Nm CSS-H138A,Nm CSS-R173A,and Nm CSS-H138A R173A.?4?The related mutant proteins were induced to express and purify in E.coli.The protease activity was determined by the method of detecting phosphate radical with malachite green.The results showed that the three mutants of Nm CSS had some decrease in sialic acid activity for natural substrates,but increased ^FITC-SA activity.Compared with wild-type Nm CSS,the three mutants increased ^FITC-SA enzyme activity by about 4-fold.Among them,the enzyme activity of Nm CSS-R173A was slightly higher than that of Nm CSS-H138A and Nm CSS-H138A R173A.In this study,we obtained the Nm CSS mutant R173A with higher recognition and affinity for ^FITC-SA by rational design and combined with experimental validation.The acquisition of this mutant lays the foundation for the further application of Nm CSS to chemo-enzymatic labeling of glycans.