16465-33-1Relevant academic research and scientific papers
Eliminations in the reactions catalyzed by UDP-N-acetylglucosamine 2-epimerase
Morgan, Paul M.,Sala, Rafael F.,Tanner, Martin E.
, p. 10269 - 10277 (1997)
Mechanistic studies have been carried out on the bacterial enzyme UDP-N-acetylglucosamine 2-epimerase, which catalyzes the interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). This enzyme is interesting because it epimerizes a stereocenter that does not bear an acidic proton, and therefore it cannot utilize a simple deprotonation/reprotonation mechanism. A coupled enzyme assay employing UDP-ManNAc dehydrogenase has been developed. The epimerization in D2O is found to be accompanied by the incorporation of deuterium into the C-2'' position of both epimers, supporting a mechanism that ultimately involves a proton transfer at this position. The epimerization of [2''-2H]UDP-GlcNAc is slowed by a primary kinetic isotope effect indicating that C-H bond cleavage is occurring during a rate-determining step of the reaction. A positional isotope exchange (PIX) experiment shows that an 18O label in the sugar-UDP bridging position will scramble into nonbridging diphosphate positions during enzymatic epimerization. These observations are consistent with a mechanism that proceeds via cleavage of the anomeric C-O bond, with 2-acetamidoglucal and UDP as enzyme-bound intermediates. Additional evidence for this mechanism is found in the unusual observation that during extended incubations, the intermediates are gradually released from the enzyme and accumulate in solution. These intermediates are formed by an anti elimination of UDP from UDP-GlcNAc and a syn elimination of UDP from UDP-ManNAc. It is likely that E1-like eliminations via oxocarbenium intermediates are involved in the reaction. Further experiments show that 3''-deoxy-UDP-GlcNAc is not a substrate for the enzyme and that the enzyme does not contain a tightly bound NAD+ cofactor.
Gram-scale production of sugar nucleotides and their derivatives
Li, Shuang,Wang, Shuaishuai,Wang, Yaqian,Qu, Jingyao,Liu, Xian-Wei,Wang, Peng George,Fang, Junqiang
supporting information, p. 2628 - 2633 (2021/04/21)
Here, we report a practical sugar nucleotide production strategy that combined a high-concentrated multi-enzyme catalyzed reaction and a robust chromatography-free selective precipitation purification process. Twelve sugar nucleotides were synthesized on a gram scale with a purity up to 98%.
Characterization and mutational analysis of two UDP-galactose 4-epimerases in Streptococcus pneumoniae TIGR4
Chen,Han,Zhai,Wang,Wang,Chen
, p. 37 - 44 (2018/02/06)
Current clinical treatments for pneumococcal infections have many limitations and are faced with many challenges. New capsular polysaccharide structures must be explored to cope with diseases caused by different serotypes of Streptococcus pneumoniae. UDP-galactose 4-epimerase (GalE) is an essential enzyme involved in polysaccharide synthesis. It is an important virulence factor in many bacterial pathogens. In this study, we found that two genes (galEsp1 and galEsp2) are responsible for galactose metabolism in pathogenic S. pneumoniae TIGR4. Both GalESp1 and GalESp2 were shown to catalyze the epimerization of UDP-glucose (UDP-Glc)/UDP-galactose (UDP-Gal), but only GalESp2 was shown to catalyze the epimerization of UDP-N-acetylglucosamine (UDP-GlcNAc)/UDP-N-acetylgalactosamine (UDP-GalNAc). Interestingly, GalESp2 had 3-fold higher epimerase activity toward UDP-Glc/UDP-Gal than GalESp1. The biochemical properties of GalESp2 were studied. GalESp2 was stable over a wide range of temperatures, between 30 and 70°C, at pH 8.0. The K86G substitution caused GalESp2 to lose its epimerase activity toward UDP-Glc and UDP-Gal; however, substitution C300Y in GalESp2 resulted in only decreased activity toward UDP-GlcNAc and UDP-GalNAc. These results indicate that the Lys86 residue plays a critical role in the activity and substrate specificity of GalESp2.
Efficient chemoenzymatic synthesis of uridine 5′-diphosphate N-acetylglucosamine and uridine 5′-diphosphate N-trifluoacetyl glucosamine with three recombinant enzymes
Li, Xiaoyan,Qi, Chen,Wei, Peilian,Huang, Lei,Cai, Jin,Xu, Zhinan
, p. 852 - 859 (2017/10/05)
Uridine 5′-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a natural UDP-monosaccharide donor for bacterial glycosyltransferases, while uridine 5′-diphosphate N-trifluoacetyl glucosamine (UDP-GlcNTFA) is its synthetic mimic. The chemoenzymatic synthesis of UDP-GlcNAc and UDP-GlcNTFA was attempted by three recombinant enzymes. Recombinant N-acetylhexosamine 1-kinase was used to produce GlcNAc/GlcNTFA-1-phosphate from GlcNAc/GlcNTFA. N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12 MG1655 was used to produce UDP-GlcNAc/GlcNTFA from GlcNAc/GlcNTFA-1-phosphate. Inorganic pyrophosphatase from E. coli K12 MG1655 was used to hydrolyze pyrophosphate to accelerate the reaction. The above enzymes were expressed in E. coli BL21 (DE3) and purified, respectively, and finally mixed in one-pot bioreactor. The effects of reaction conditions on the production of UDP-GlcNAc and UDP-GlcNTFA were characterized. To avoid the substrate inhibition effect on the production of UDP-GlcNAc and UDP-GlcNTFA, the reaction was performed with fed batch of substrate. Under the optimized conditions, high production of UDP-GlcNAc (59.51 g/L) and UDP-GlcNTFA (46.54 g/L) were achieved in this three-enzyme one-pot system. The present work is promising to develop an efficient scalable process for the supply of UDP-monosaccharide donors for oligosaccharide synthesis.
Probing the roles of conserved residues in uridyltransferase domain of Escherichia coli K12 GlmU by site-directed mutagenesis
Wang, Shuaishuai,Fu, Xuan,Liu, Yunpeng,Liu, Xian-Wei,Wang, Lin,Fang, Junqiang,Wang, Peng George
supporting information, p. 70 - 74 (2015/06/30)
N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme that catalyzes both acetyltransfer and uridyltransfer reactions in the prokaryotic UDP-GlcNAc biosynthesis pathway. Our previous study demonstrated that the uridyltransferase domain of GlmU (tGlmU) exhibited a flexible substrate specificity, which could be further applied in unnatural sugar nucleotides preparation. However, the structural basis of tolerating variant substrates is still not clear. Herein, we further investigated the roles of several highly conserved amino acid residues involved in substrate binding and recognition by structure- and sequence-guided site-directed mutagenesis. Out of total 16 mutants designed, tGlmU Q76E mutant which had a novel catalytic activity to convert CTP and GlcNAc-1P into unnatural sugar nucleotide CDP-GlcNAc was identified. Furthermore, tGlmU Y103F and N169R mutants were also investigated to have enhanced uridyltransferase activities compared with wide-type tGlmU.
Biosynthesis of the carbamoylated D-gulosamine moiety of streptothricins: Involvement of a guanidino-N-glycosyltransferase and an N-acetyl-D-gulosamine deacetylase
Guo, Zhengyan,Li, Jine,Qin, Hua,Wang, Min,Lv, Xun,Li, Xuebing,Chen, Yihua
, p. 5175 - 5178 (2015/04/27)
Streptothricins (STNs) are atypical aminoglycosides containing a rare carbamoylated D-gulosamine (D-GulN) moiety, and the antimicrobial activity of STNs has been exploited for crop protection. Herein, the biosynthetic pathway of the carbamoylated D-GulN moiety was delineated. An N-acetyl-D-galactosamine is first attached to the streptolidine lactam by the glycosyltransferse StnG and then epimerized to N-acetyl-D-gulosamine by the putative epimerase StnJ. After carbamoylation by the carbamoyltransferase StnQ, N-acetyl-D-GulN is deacetylated by StnI to furnish the carbamoylated D-GulN moiety. In vitro studies characterized two novel enzymes: StnG is an unprecedented GT-A fold N-glycosyltransferase that glycosylates the imine nitrogen atom of guanidine, and StnI is the first reported N-acetyl-D-GulN deacetylase. The dynamic duo: Two novel enzymes, StnG and StnI, have been found to be involved in the biosynthetic pathway of the carbamoylated D-gulosamine moiety in streptothricins. StnG is a GT-A fold glycosyltransferase that catalyzes the unprecedented attachment of a sugar to the imine nitrogen atom of a guanidine group; StnI catalyzes the deacetylation of the N-acetyl-D-gulosamine moiety.
Enzymatic synthesis of nucleobase-modified UDP-sugars: Scope and limitations
Wagstaff, Ben A.,Rejzek, Martin,Pesnot, Thomas,Tedaldi, Lauren M.,Caputi, Lorenzo,O'Neill, Ellis C.,Benini, Stefano,Wagner, Gerd K.,Field, Robert A.
, p. 17 - 25 (2015/03/05)
Glucose-1-phosphate uridylyltransferase in conjunction with UDP-glucose pyrophosphorylase was found to catalyse the conversion of a range of 5-substituted UTP derivatives into the corresponding UDP-galactose derivatives in poor yield. Notably the 5-iodo derivative was not converted to UDP-sugar. In contrast, UDP-glucose pyrophosphorylase in conjunction with inorganic pyrophosphatase was particularly effective at converting 5-substituted UTP derivatives, including the iodo compound, into a range of gluco-configured 5-substituted UDP-sugar derivatives in good yields. Attempts to effect 4″-epimerization of these 5-substituted UDP-glucose with UDP-glucose 4″-epimerase from yeast were unsuccessful, while use of the corresponding enzyme from Erwinia amylovora resulted in efficient epimerization of only 5-iodo-UDP-Glc, but not the corresponding 5-aryl derivatives, to give 5-iodo-UDP-Gal. Given the established potential for Pd-mediated cross-coupling of 5-iodo-UDP-sugars, this provides convenient access to the galacto-configured 5-substituted-UDP-sugars from gluco-configured substrates and 5-iodo-UTP.
CHEMOENZYMATIC SYNTHESIS OF HEPARIN AND HEPARAN SULFATE ANALOGS
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Paragraph 0323; 0324; 0329, (2014/09/03)
The present invention provides a one-pot multi-enzyme method for preparing UDP-sugars from simple sugar starting materials. The invention also provides a one-pot multi-enzyme method for preparing oligosaccharides from simple sugar starting materials.
Sequential one-pot enzymatic synthesis of oligo-N-acetyllactosamine and its multi-sialylated extensions
Chien, Wei-Ting,Liang, Chien-Fu,Yu, Ching-Ching,Lin, Chien-Hung,Li, Si-Peng,Primadona, Indah,Chen, Yu-Ju,Mong, Kwok Kong T.,Lin, Chun-Cheng
supporting information, p. 5786 - 5789 (2014/05/20)
A simple and efficient protocol for the preparative-scale synthesis of various lengths of oligo-N-acetyllactosamine (oligo-LacNAc) and its multi-sialylated extensions is described. The strategy utilizes one thermophilic bacterial thymidylyltransferase (RmlA) coupled with corresponding sugar-1-phosphate kinases to generate two uridine diphosphate sugars, UDP-galactose and UDP-N-acetylglucosamine. By incorporating glycosyltransferases, oligo-LacNAcs and their sialylated analogs were synthesized. the Partner Organisations 2014.
A chemoenzymatic route to synthesize unnatural sugar nucleotides using a novel N-acetylglucosamine-1-phosphate pyrophosphorylase from Camphylobacter jejuni NCTC 11168
Fang, Junqiang,Xue, Mengyang,Gu, Guofeng,Liu, Xian-Wei,Wang, Peng George
supporting information, p. 4303 - 4307 (2013/07/26)
A novel N-acetylglucosamine-1-phosphate pyrophosphorylase was identified from Campylobacter jejuni NCTC 11168. An unprecedented degree of substrate promiscuity has been revealed by systematic studies on its substrate specificities towards sugar-1-P and NTP. The yields of the synthetic reaction of seven kinds of sugar nucleotides catalyzed by the enzyme were up to 60%. In addition, the yields of the other nine were around 20%. With this enzyme, three novel sugar nucleotide analogs were synthesized on a preparative scale and well characterized.
