Structure,Function And Molecular Modification Of Key Enzymes In Artificial Synthesis Pathway Of Glycosphingolipids

Glycosphingolipids?GSLs?are a class of amphipathic compounds composed of a hydrophilic oligosaccharides head group moiety and a common hydrophobic ceramide moiety on the cell surface of all vertebrates.They are involved in a range of physiological and pathological processes as a class of bioactive molecule,such as cellular signaling,immunology,cancer,and insulin resistance.Therefore,GSLs have potential as diagnostic markers,therapeutic targets or drugs for various diseases,and have attracted extensive attention in pharmaceutical field.Isolation of GSLs from natural sources is common but subject to several notable limitations,including potential transmission of disease,heterogeneity,and,in some cases,scarcity.Chemical synthesis,albeit possible,remains laborious and poor yielding given the need for multiple activation and protection steps to achieve regio-and stereo-selectivity.Using synthetic biology techniques to construct artificial synthetic pathway of GSLs through systemic design rationally,will contribute to the achievement of the productive and innovative GSLs-derived therapeutics.In previous work in our laboratory,we have initially established a multi-enzyme synthetic system of GSLs-derived therapeutics based on the concept of in vitro synthetic biology.In order to further improve the efficiency of artificial system and finally realize the production of GSLs in large-scale,we have focused on three key catalytic enzymes in the artificial system,including endoglycoceramidase?EGCase?,sphingolipid ceramide N-deacylase?SCDase?and fucosyltransferase?FucT?.We did systematic study about them by structure-based mechanism analysis,characterization of enzymatic properties and enzyme engineering.Endoglycoceramidase?EGCase?is a glycosidase capable of hydrolyzing the?-glycosidic linkage between the oligosaccharides and ceramides of GSLs.By mutating the nucleophilic glutamate to a non-nucleophilic residue of EGCase,the glycosidase was rendered incapable of hydrolyzing GSLs,but could catalyze their formation from the appropriate sugar?-fluoride donor and sphingosine to generate lyso-GSLs.We identify a novel EGCase with broad specificity and good reaction efficiency from Rhodococcus equi 103S,which can be readily and functionally expressed in E.coli.The k_cat/K_m of 103S_EGC I for GM1 is 130-fold higher than that of a widely used EGCase II from Rhodococcus sp.M-777.In order to elucidate the mechanism of substrate specificity of EGCase,we resolve the x-ray crystal structures of apo-EGCase I and the complex of ganglioside substrate GM1 with a hydrolytically inactive nucleophile mutant.The substrate binding sites were analyzed and compared with those of EGCase II in detail.We identify some key residues that affect the activity of EGCase II,and we get a good mutant with 10-folds activity enhancement and broader substrate specificity.This study represents a significant advance in our understanding of the substrate specificity of EGCase,and further offer valuable information for rational redesign of EGCase I toward the synthesis of novel GSLs-derived therapeutics.Sphingolipid ceramide N-deacylase?SCDase?catalyzes reversible reactions in which the amide linkage in glycosphingolipids is hydrolyzed or synthesized.While SCDases show great value for the enzymatic synthesis of glycosphingolipids,they are relatively poorly characterized enzymes.In this work,the enzymatic properties of SCDase from Shewanella alga G8?SA_SCD?were systematically characterized and compared with the commercially available SCDase from Pseudomonas sp.TK4?PS_SCD?.The optimal pH values for the hydrolytic and synthetic activity of SA_SCD were pH 6.0 and pH 7.5,respectively.Both activities were strongly inhibited by Zn²⁺and Cu²⁺,while Fe²⁺,Co²⁺,Ni²⁺,Mn²⁺,Ca²⁺and Mg²⁺promoted the hydrolytic activity but inhibited the synthetic activity.SA_SCD showed very broad substrate specificity both in hydrolysis and synthesis.Importantly,SA_SCD has a broader specificity for acyl donors acceptance than does PS_SCD,especially for unsaturated fatty acids and fatty acids with very short or long acyl chains.Further kinetic analysis revealed that the k_cat/K_m value for the hydrolytic activity of SA_SCD was 9-fold higher than that of PS_SCD for GM1,while the values for the synthetic activity were 38-fold higher for stearic acid and 23-fold higher for lyso-GM1?d18:1?than those of PS_SCD,respectively.The broad fatty acid specificity and high catalytic efficiency,together with the ease of expression of SA_SCD in Escherichia coli,make it a better biocatalyst than is PS_SCD for the synthesis and structural remodelling of GSLs.Fucosylation is involved in a variety of biological and pathological processes in eukaryotic organisms including tissue development,angiogenesis,fertilization,cell adhesion,inflammation,and tumor metastasis.Fucosyltransferases?FucTs?are the enzymes responsible for the catalysis of fucose transfer from GDP-fucose to various acceptor molecules.FucTs have significant applied interest for the enzymatic and microbial synthesis of GSLs-derived therapeutic cancer vaccine and functional ingredients.However,the low activity and strict substrate specificities of many kinds of FucTs limit their practical application.Here,we report the development of a new fluorescence-based cell sorting?FACS?high-throughput screening methodology for the directed evolution of FucTs,with ability to analyze as many as 10⁷mutants/h,using the?1,3-fucosyltransferase?FutA?from H.pylori NCTC11639 as a model enzyme.In the model screening,the E.coli cells expressing the wild-type FutA can be enriched up to 10000 folds from a mixture sample containing a large number of cells lacking the enzyme?empty vector transformants?.Moreover,this method can distinguish cells with 2-fold activity difference.In a model quantitative screening experiment,the higher activity mutant can be enriched 6-20 folds in different reference mix ratio.Then,we generate a huge library of FutA mutants?⁴×10⁶?by error-prone PCR with?2-5 mutations per gene.A three rounds screening strategy based FACS methods was used for the FutA random library screen.Some mutants with higher activity were significantly enriched.Finally,we get some mutants with a significant activity enhancement,such as A4?Y199N/V368A/D407N?and D9?E340D?,with 30%and 70%activity enhancement respectively.In addition to the?1,3-FucT,we have shown that this E.coli-based screening system can also be readily applied to?1,2-FucT by designing suitable fluorescence-labeled acceptor,thus expand the range of application and lay the foundation for broader engineering of FucT.In summary,this doctoral dissertation did systematic study for the key enzymes of artificial multi-enzyme synthetic system of GSLs.We found the new and good enzymes of EGCase and SCDase,elucidated the mechanism of substrate selectivity of EGCase,developed a high-throughput screening methods for FucT,and got a variety of improved mutants by enzyme engineering.This work will further help design and optimize the artificial synthetic system of GSLs,and ultimately to provide technical support and the theory origin for efficient synthesis and new structure generation of GSLs-derived therapeutics.