- Gram-scale production of sugar nucleotides and their derivatives
-
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%.
- Li, Shuang,Wang, Shuaishuai,Wang, Yaqian,Qu, Jingyao,Liu, Xian-Wei,Wang, Peng George,Fang, Junqiang
-
supporting information
p. 2628 - 2633
(2021/04/21)
-
- Characterization and mutational analysis of two UDP-galactose 4-epimerases in Streptococcus pneumoniae TIGR4
-
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.
- Chen,Han,Zhai,Wang,Wang,Chen
-
-
- Enzymatic Synthesis of Human Milk Fucosides α1,2-Fucosyl para-Lacto-N-Hexaose and its Isomeric Derivatives
-
Enzymatic synthesis of para-lacto-N-hexaose and its isomeric structures as well as those α1,2-fucosylated variants naturally occurring in human milk oligosaccharide (HMOs) was achieved using a sequential one-pot enzymatic system. Three glycosylation routes comprising bacterial glycosyltransferases and corresponding sugar-nucleotide-generating enzymes were developed to facilitate efficient production of extended type-1 and type-2 N-acetyllactosamine (LacNAc) backbones and hybrid chains. Further fucosylation efficiency of two α1,2-fucosyltransferases on both type-1 and type-2 chains of the hexasaccharide was investigated to achieve practical synthesis of the fucosylated glycans. The availability of structurally defined HMOs offers a practical approach for investigating future biological applications. (Figure presented.).
- Fang, Jia-Lin,Tsai, Teng-Wei,Liang, Chin-Yu,Li, Jyun-Yi,Yu, Ching-Ching
-
p. 3213 - 3219
(2018/08/06)
-
- Efficient chemoenzymatic synthesis of uridine 5′-diphosphate N-acetylglucosamine and uridine 5′-diphosphate N-trifluoacetyl glucosamine with three recombinant enzymes
-
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.
- Li, Xiaoyan,Qi, Chen,Wei, Peilian,Huang, Lei,Cai, Jin,Xu, Zhinan
-
p. 852 - 859
(2017/10/05)
-
- Probing the roles of conserved residues in uridyltransferase domain of Escherichia coli K12 GlmU by site-directed mutagenesis
-
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.
- Wang, Shuaishuai,Fu, Xuan,Liu, Yunpeng,Liu, Xian-Wei,Wang, Lin,Fang, Junqiang,Wang, Peng George
-
supporting information
p. 70 - 74
(2015/06/30)
-
- Biosynthesis of the carbamoylated D-gulosamine moiety of streptothricins: Involvement of a guanidino-N-glycosyltransferase and an N-acetyl-D-gulosamine deacetylase
-
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.
- Guo, Zhengyan,Li, Jine,Qin, Hua,Wang, Min,Lv, Xun,Li, Xuebing,Chen, Yihua
-
p. 5175 - 5178
(2015/04/27)
-
- Enzymatic synthesis of nucleobase-modified UDP-sugars: Scope and limitations
-
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.
- Wagstaff, Ben A.,Rejzek, Martin,Pesnot, Thomas,Tedaldi, Lauren M.,Caputi, Lorenzo,O'Neill, Ellis C.,Benini, Stefano,Wagner, Gerd K.,Field, Robert A.
-
-
- CHEMOENZYMATIC SYNTHESIS OF HEPARIN AND HEPARAN SULFATE ANALOGS
-
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.
- -
-
Paragraph 0323; 0324; 0329
(2014/09/03)
-
- Sequential one-pot enzymatic synthesis of oligo-N-acetyllactosamine and its multi-sialylated extensions
-
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.
- 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)
-
- One-pot three-enzyme synthesis of UDP-Glc, UDP-Gal, and their derivatives
-
A UTP-glucose-1-phosphate uridylyltransferase (SpGalU) and a galactokinase (SpGalK) were cloned from Streptococcus pneumoniae TIGR4 and were successfully used to synthesize UDP-galactose (UDP-Gal), UDP-glucose (UDP-Glc), and their derivatives in an efficient one-pot reaction system. The reaction conditions for the one-pot multi-enzyme synthesis were optimized and nine UDP-Glc/Gal derivatives were synthesized. Using this system, six unnatural UDP-Gal derivatives, including UDP-2-deoxy-Galactose and UDP-GalN3 which were not accepted by other approach, can be synthesized efficiently in a one pot fashion. More interestingly, this is the first time it has been reported that UDP-Glc can be synthesized in a simpler one-pot three-enzyme synthesis reaction system.
- Zou, Yang,Xue, Mengyang,Wang, Wenjun,Cai, Li,Chen, Leilei,Liu, Jun,Wang, Peng George,Shen, Jie,Chen, Min
-
supporting information
p. 76 - 81
(2013/06/27)
-
- A novel allosteric inhibitor of the uridine diphosphate N-acetylglucosamine pyrophosphorylase from Trypanosoma brucei
-
Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final reaction in the biosynthesis of UDP-GlcNAc, an essential metabolite in many organisms including Trypanosoma brucei, the etiological agent of Human African Trypanosomiasis. High-throughput screening of recombinant T. brucei UAP identified a UTP-competitive inhibitor with selectivity over the human counterpart despite the high level of conservation of active site residues. Biophysical characterization of the UAP enzyme kinetics revealed that the human and trypanosome enzymes both display a strictly ordered bi-bi mechanism, but with the order of substrate binding reversed. Structural characterization of the T. brucei UAP-inhibitor complex revealed that the inhibitor binds at an allosteric site absent in the human homologue that prevents the conformational rearrangement required to bind UTP. The identification of a selective inhibitory allosteric binding site in the parasite enzyme has therapeutic potential.
- Urbaniak, Michael D.,Collie, Iain T.,Fang, Wenxia,Aristotelous, Tonia,Eskilsson, Susanne,Raimi, Olawale G.,Harrison, Justin,Navratilova, Iva Hopkins,Frearson, Julie A.,Van Aalten, Daan M. F.,Ferguson, Michael A. J.
-
p. 1981 - 1987
(2013/10/08)
-
- A chemoenzymatic route to synthesize unnatural sugar nucleotides using a novel N-acetylglucosamine-1-phosphate pyrophosphorylase from Camphylobacter jejuni NCTC 11168
-
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.
- Fang, Junqiang,Xue, Mengyang,Gu, Guofeng,Liu, Xian-Wei,Wang, Peng George
-
supporting information
p. 4303 - 4307
(2013/07/26)
-
- Biosynthesis of nucleotide sugars by a promiscuous UDP-sugar pyrophosphorylase from Arabidopsis thaliana (AtUSP)
-
Nucleotide sugars are activated forms of monosaccharides and key intermediates of carbohydrate metabolism in all organisms. The availability of structurally diverse nucleotide sugars is particularly important for the characterization of glycosyltransferases. Given that limited methods are available for preparation of nucleotide sugars, especially their useful non-natural derivatives, we introduced herein an efficient one-step three-enzyme catalytic system for the synthesis of nucleotide sugars from monosaccharides. In this study, a promiscuous UDP-sugar pyrophosphorylase (USP) from Arabidopsis thaliana (AtUSP) was used with a galactokinase from Streptococcus pneumoniae TIGR4 (SpGalK) and an inorganic pyrophosphatase (PPase) to effectively synthesize four UDP-sugars. AtUSP has better tolerance for C4-derivatives of Gal-1-P compared to UDP-glucose pyrophosphorylase from S. pneumoniae TIGR4 (SpGalU). Besides, the nucleotide substrate specificity and kinetic parameters of AtUSP were systematically studied. AtUSP exhibited considerable activity toward UTP, dUTP and dTTP, the yield of which was 87%, 85% and 84%, respectively. These results provide abundant information for better understanding of the relationship between substrate specificity and structural features of AtUSP.
- Liu, Jun,Zou, Yang,Guan, Wanyi,Zhai, Yafei,Xue, Mengyang,Jin, Lan,Zhao, Xueer,Dong, Junkai,Wang, Wenjun,Shen, Jie,Wang, Peng George,Chen, Min
-
supporting information
p. 3764 - 3768
(2013/07/25)
-
- Biosynthesis of the tunicamycin antibiotics proceeds via unique exo-glycal intermediates
-
The tunicamycins are archetypal nucleoside antibiotics targeting bacterial peptidoglycan biosynthesis and eukaryotic protein N-glycosylation. Understanding the biosynthesis of their unusual carbon framework may lead to variants with improved selectivity. Here, we demonstrate in vitro recapitulation of key sugar-manipulating enzymes from this pathway. TunA is found to exhibit unusual regioselectivity in the reduction of a key α,β-unsaturated ketone. The product of this reaction is shown to be the preferred substrate for TunF-an epimerase that converts the glucose derivative to a galactose. In Streptomyces strains in which another gene (tunB) is deleted, the biosynthesis is shown to stall at this exo-glycal product. These investigations confirm the combined TunA/F activity and delineate the ordering of events in the metabolic pathway. This is the first time these surprising exo-glycal intermediates have been seen in biology. They suggest that construction of the aminodialdose core of tunicamycin exploits their enol ether motif in a mode of C-C bond formation not previously observed in nature, to create an 11-carbon chain.
- Wyszynski, Filip J.,Lee, Seung Seo,Yabe, Tomoaki,Wang, Hua,Gomez-Escribano, Juan Pablo,Bibb, Mervyn J.,Lee, Soo Jae,Davies, Gideon J.,Davis, Benjamin G.
-
experimental part
p. 539 - 546
(2012/09/08)
-
- Metabolism of vertebrate amino sugars with N-glycolyl groups: Intracellular β-O-linked N-glycolylglucosamine (GlcNGc), UDP-GlcNGc,and the biochemical and structural rationale for the substrate tolerance of β-O-linked& β-N-acetylglucosaminidase
-
The O-GlcNAc modification involves the attachment of single β-O-linked N-acetylglucosamine residues to serine and threonine residues of nucleocytoplasmic proteins. Interestingly, previous biochemical and structural studies have shown that O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from proteins, has an active site pocket that tolerates various N-acyl groups in addition to the N-acetyl group of GlcNAc. The remarkable sequence and structural conservation of residues comprising this pocket suggest functional importance. We hypothesized this pocket enables processing of metabolic variants of O-GlcNAc that could be formed due to inaccuracy within the metabolic machinery of the hexosamine biosynthetic pathway. In the accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, 28865-28881), N-glycolylglucosamine (GlcNGc) was shown to be a catabolite of NeuNGc. Here, we show that the hexosamine salvage pathway can convert GlcNGc to UDP-GlcNGc, which is then used to modify proteins with O-GlcNGc. The kinetics of incorporation and removal of O-GlcNGc in cells occur in a dynamic manner on a time frame similar to that of O-GlcNAc. Enzymatic activity of O-GlcNAcase (OGA) toward a GlcNGc glycoside reveals OGA can process glycolyl-containing substrates fairly efficiently. A bacterial homolog (BtGH84) of OGA, from a human gut symbiont, also processes O-GlcNGc substrates, and the structure of this enzyme bound to a GlcNGc-derived species reveals the molecular basis for tolerance and binding of GlcNGc. Together, these results demonstrate that analogs of GlcNAc, such as GlcNGc, are metabolically viable species and that the conserved active site pocket of OGA likely evolved to enable processing of mis-incorporated analogs of O-GlcNAc and thereby prevent their accumulation. Such plasticity in carbohydrate processing enzymes may be a general feature arising from inaccuracy in hexosamine metabolic pathways.
- Macauley, Matthew S.,Chan, Jefferson,Zandberg, Wesley F.,He, Yuan,Whitworth, Garrett E.,Stubbs, Keith A.,Yuzwa, Scott A.,Bennet, Andrew J.,Varki, Ajit,Davies, Gideon J.,Vocadlo, David J.
-
p. 28882 - 28897
(2012/11/07)
-
- Efficient one-pot multienzyme synthesis of UDP-sugars using a promiscuous UDP-sugar pyrophosphorylase from Bifidobacterium longum (BLUSP)
-
A promiscuous UDP-sugar pyrophosphorylase (BLUSP) was cloned from Bifidobacterium longum strain ATCC55813 and used efficiently with a Pasteurella multocida inorganic pyrophosphatase (PmPpA) with or without a monosaccharide 1-kinase for one-pot multienzyme synthesis of UDP-galactose, UDP-glucose, UDP-mannose, and their derivatives. Further chemical diversification of a UDP-mannose derivative resulted in the formation of UDP-N-acetylmannosamine. The Royal Society of Chemistry 2012.
- Muthana, Musleh M.,Qu, Jingyao,Li, Yanhong,Zhang, Lei,Yu, Hai,Ding, Li,Malekan, Hamed,Chen, Xi
-
p. 2728 - 2730
(2012/04/17)
-
- Investigation of the nucleotide triphosphate substrate specificity of Homo sapiens UDP-N-acetylgalactosamine pyrophosphorylase (AGX1)
-
Nucleotide sugars are essential glycosyl donors for Leloir-type glycosyltransferases. The UDP-N-acetylgalactosamine pyrophosphorylase (UDP-GalNAc PP; AGX1) from Homo sapiens catalyzes the synthesis of UDP-N-acetylgalactosamine from N-acetylgalactosamine 1-phosphate and UTP. In this Letter, we systematically studied nucleotide substrate specificity of AGX1 during its uridyltransfer reaction, and described the capability of AGX1 to catalyze dUTP and dTTP to their corresponding nucleotide sugars for the first time. Furthermore, using such a eukaryotic enzyme, we synthesized dUDP-GalNAc and dTDP-GalNAc in multiple mg scale in vitro efficiently and rapidly.
- Xue, Mengyang,Guan, Wanyi,Zou, Yang,Fang, Junqiang,Liu, Xian-Wei,Wang, Peng George,Wang, Fengshan
-
scheme or table
p. 3957 - 3961
(2012/07/03)
-
- One-pot three-enzyme synthesis of UDP-GlcNAc derivatives
-
A Pasteurella multocida N-acetylglucosamine 1-phosphate uridylyltransferase (PmGlmU) was cloned and used efficiently with an N-acetylhexosamine 1-kinase (NahK-ATCC55813) and an inorganic pyrophosphatase (PmPpA) for one-pot three-enzyme synthesis of UDP-GlcNAc derivatives with or without further chemical diversification.
- Chen, Yi,Thon, Vireak,Li, Yanhong,Yu, Hai,Ding, Li,Lau, Kam,Qu, Jingyao,Hie, Liana,Chen, Xi
-
supporting information; experimental part
p. 10815 - 10817
(2011/11/04)
-
- Characterization of a bifunctional pyranose-furanose mutase from Campylobacter jejuni 11168
-
UDP-galactopyranose mutases (UGM) are the enzymes responsible for the synthesis of UDP-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme, encoded by the glf gene, is present in bacteria, parasites, and fungi that express Galf in their glycoconjugates. Recently, a UGM homologue encoded by the cj1439 gene has been identified in Campylobacter jejuni 11168, an organism possessing no Galf-containing glycoconjugates. However, the capsular polysaccharide from this strain contains a 2-acetamido-2-deoxy-D-galactofuranose (GalfNAc) moiety. Using an in vitro high performance liquid chromatography assay and complementation studies, we characterized the activity of this UGM homologue. The enzyme, which we have renamed UDP-N-acetylgalactopyranose mutase (UNGM), has relaxed specificity and can use either UDP-Gal or UDP-GalNAc as a substrate. Complementation studies of mutase knock-outs in C. jejuni 11168 and Escherichia coli W3110, the latter containing Galf residues in its lipopolysaccharide, demonstrated that the enzyme recognizes both UDP-Gal and UDP-GalNAc in vivo. A homology model of UNGM and site-directed mutagenesis led to the identification of two active site amino acid residues involved in the recognition of the UDP-GalNAc substrate. The specificity of UNGM was characterized using a two-substrate co-incubation assay, which demonstrated, surprisingly, that UDP-Gal is a better substrate than UDP-GalNAc.
- Poulin, Myles B.,Nothaft, Harald,Hug, Isabelle,Feldman, Mario F.,Szymanski, Christine M.,Lowary, Todd L.
-
experimental part
p. 493 - 501
(2010/11/18)
-
- Identification and characterization of a strict and a promiscuous N-acetylglucosamine-1-P uridylyltransferase in Arabidopsis
-
UDP-GlcNAc is an essential precursor for glycoprotein and glycolipid synthesis. In the present study, a functional nucleotidyltransferase gene from Arabidopsis encoding a 58.3 kDa GlcNAc1pUT-1 (N-acetylglucosamine-1-phosphate uridylyltransferase) was identified. In the forward reaction the enzyme catalyses the formation of UDP-N-acetylglucosamine and PPi from the respective monosaccharide 1-phosphate and UTP. The enzyme can utilize the 4-epimer UDP-GalNAc as a substrate as well. The enzyme requires divalent ions (Mg2+ or Mn2+) for activity and is highly active between pH 6.5 and 8.0, and at 30-37°C. The apparent Km values for the forward reaction were 337 μM (GlcNAc-1-P) and 295 μM (UTP) respectively. Another GlcNAc1pUT-2, which shares 86%amino acid sequence identity with GlcNAc1pUT-1, was found to convert, in addition to GlcNAc-1-P and GalNAc-1-P, Glc-1-P into corresponding UDP-sugars, suggesting that subtle changes in the UT family cause different substrate specificities. A three-dimensional protein structure model using the human AGX1 as template showed a conserved catalytic fold and helped identify key conserved motifs, despite the high sequence divergence. The identification of these strict and promiscuous gene products open a window to indentify new roles of amino sugar metabolism in plants and specifically their role as signalling molecules. The ability of GlcNAc1pUT-2 to utilize three different substrates may provide further understanding as to why biological systems have plasticity. The Authors.
- Yang, Ting,Echols, Merritt,Martin, Andy,Bar-Peled, Maor
-
experimental part
p. 275 - 284
(2012/05/20)
-
- Methods and Compositions for Modulating Glycosylation
-
The invention relates to methods and products for modulating glycosylation of proteins. The invention is useful for treating glycosylation-associated disorders such as neurodegeneration, diabetes, including complications of diabetes such as insulin resistance, nephropathy, microvascular damage, and endothelial dysfunction. The invention also relates in part to assays that are useful for identifying and testing candidate compounds for modulating glycosylation of proteins.
- -
-
-
- Enzymatic synthesis of UDP-GlcNAc/UDP-GalNAc analogs using N-acetylglucosamine 1-phosphate uridyltransferase (GlmU)
-
Reports the generation of a library composed of UDP-GlcNAc/UDP-GalNAc and investigates the substrate specificity of Escherichia coli GlcNAc-1-P uridyltransferase GlmU. The Royal Society of Chemistry 2009.
- Guan, Wanyi,Cai, Li,Fang, Junqiang,Wu, Baolin,George Wang, Peng
-
experimental part
p. 6976 - 6978
(2010/04/25)
-
- Systematic study on the broad nucleotide triphosphate specificity of the pyrophosphorylase domain of the N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12
-
N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) from Escherichia coli K12 is a bifunctional enzyme that catalyzes both the acetyltransfer and uridyltransfer reactions in the prokaryotic UDP-GlcNAc biosynthetic pathway. In this study, we report th
- Fang, Junqiang,Guan, Wanyi,Cai, Li,Gu, Guofeng,Liu, Xianwei,Wang, Peng George
-
supporting information; experimental part
p. 6429 - 6432
(2010/06/11)
-
- Exploiting nucleotidylyltransferases to prepare sugar nucleotides
-
(Graph Presented) Enzymatic approaches to prepare sugar nucleotides are gaining in importance and offer several advantages over chemical synthesis including high yields and stereospecificity. We report the cloning, expression, and purification of two new wild-type thymidylyltransferases and observed catalysis with a wide variety of substrates. Significant product inhibition was not observed with the enzymes studied over a 24 h period, enabling the efficient preparation of 15 sugar nucleotides, clearly demonstrating the synthetic utility of these biocatalysts.
- Timmons, Shannon C.,Mosher, Roy H.,Knowles, Sheryl A.,Jakeman, David L.
-
p. 857 - 860
(2007/10/03)
-
- One-step synthesis of labeled sugar nucleotides for protein O-GlcNAc modification studies by chemical function analysis of an archaeal protein
-
Herein we present the chemical function analysis of a recombinant sugar nucleotidyltransferase from the hyperthermophile Pyrococcus furiosus and its use in the one-pot synthesis of chloroacetyl- and alkyne-tagged analogues of uridinediphospho-N-acetylglucosamine (UDP-GlcNAc). The gene was originally annotated as a glucose-1-phosphate deoxythymidylyltransferase; however, kinetic analysis of a panel of sugar-1-phosphates with the protein shows that it is better described as a bifunctional protein that synthesizes UDP-GlcNAc from glucosamine-1-phosphate and acetyl coenzyme A (CoA). A new mass-spectrometry-based assay for the rapid analysis of the acyltransferase activity demonstrates that the enzyme can also accept cheaper truncated N-acetylcysteamine thioester substrates in place of the natural acetyl CoA. The enzyme can tolerate alkyne or chloride substitutions in the acyl moiety, thereby allowing the facile synthesis of tagged sugar nucleotides for future use in protein O-GlcNAc modification studies. Copyright
- Mizanur, Rahman M.,Jaipuri, Firoz A.,Pohl, Nicola L.
-
p. 836 - 837
(2007/10/03)
-
- Unusually broad substrate tolerance of a heat-stable archaeal sugar nucleotidyltransferase for the synthesis of sugar nucleotides
-
Herein, we report the first cloning, recombinant expression, and synthetic utility of a sugar nucleotidyltransferase from any archaeal source and demonstrate by an electrospray ionization mass spectrometry (ESI-MS)-based assay its unusual tolerance of heat, pH, and sugar substrates. The metalion-dependent enzyme from Pyrococcus furiosus DSM 3638 showed a relatively high degree of acceptance of glucose-1-phosphate (Glc1P), mannose-1-phosphate (Man1P), galactose-1-phosphate (Gal1P), fucose-1-phosphate, glucosamine-1-phosphate, galactosamine-1-phosphate, and N-acetylglucosamine-1-phosphate with uridine and deoxythymidine triphosphate (UTP and dTTP, respectively). The apparent Michaelis constants for Glc1P, Man1P, and Gal1P are 13.0 ± 0.7, 15 ± 1, and 22 ± 2 μM, respectively, with corresponding turnover numbers of 2.08, 1.65, and 1.32 s-1, respectively. An initial velocity study indicated an ordered bi-bi catalytic mechanism for this enzyme. The temperature stability and inherently broad substrate tolerance of this archaeal enzyme promise an effective reagent for the rapid chemoenzymatic synthesis of a range of natural and unnatural sugar nucleotides for in vitro glycosylation studies and highlight the potential of archaea as a source of new enzymes for synthesis.
- Mizanur, Rahman M.,Zea, Corbin J.,Pohl, Nicola L.
-
p. 15993 - 15998
(2007/10/03)
-
- Synthesis of UDP-GalNAc analogues as probes for the study of polypeptide-α-GalNAc-transferases. Part 2
-
The synthesis of four UDP-GalNAc analogues (1-4) is described. The 3-, 4- and 6-deoxygenated analogues 1-3 were obtained by way of a divergent strategy starting from a 3,6-di-O-pivaloyl GlcNAc derivative as a common precursor. Analogue 4 bearing a N-trifluoroacetamido group was prepared from the trifluoromethylated oxazoline 24 as key intermediate. These compounds were designed to probe the substrate specificity of polypeptide-α-GalNAc transferases.
- Busca, Patricia,Martin, Olivier R.
-
p. 4433 - 4436
(2007/10/03)
-
- Active-site engineering of nucleotidylyltransferases and general enzymatic methods for the synthesis of natural and "unnatural" UDP- and TDP-nucleotide sugars
-
The present invention provides mutant nucleotidylyl-transferases, such as Ep, having altered substrate specificity; methods for their production; and methods of producing nucleotide sugars, which utilize these nucleotidylyl-transferases. The present invention also provides methods of synthesizing desired nucleotide sugars using natural and/or modified Ep or other nucleotidyltransferases; and nucleotide sugars sythesized by the present methods. The present invention further provides new glycosyl phosphates, and methods for making them.
- -
-
-
- Biocatalytic synthesis of uridine 5′-diphosphate N-acetylglucosamine by multiple enzymes co-immobilized on agarose beads
-
Recombinant N-acetylglucosamine kinase, pyruvate kinase, N-acetylglucosamine phosphate mutase, uridine 5′-diphosphate N-acetylglucosamine pyrophosphorylase, and inorganic pyrophosphatase were overexpressed in E. coli and co-immobilized on agarose beads for the practical synthesis of uridine 5′-diphosphate N-acetylglucosamine.
- Shao, Jun,Zhang, Jianbo,Nahalka, Jozef,Wang, Peng George
-
p. 2586 - 2587
(2007/10/03)
-
- Eliminations in the reactions catalyzed by UDP-N-acetylglucosamine 2-epimerase
-
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.
- Morgan, Paul M.,Sala, Rafael F.,Tanner, Martin E.
-
p. 10269 - 10277
(2007/10/03)
-
- Chemoenzymatic synthesis of UDP-N-acetyl-α-D-galactosamine
-
A novel chemoenzymatic synthesis of UDP-N-acetyl-α-D-galactosamine starting from uridine 5'-monophosphate (UMP) and sucrose is reported. In an enzymatic repetitive batch mode UDP-glucose was generated in situ from UMP and sucrose by the combination of nucleoside monophosphate kinase (EC 2.7.7.4) and sucrose synthase (EC 2.4.1.13). The transfer of UMP from UDP- glucose by galactose-1-phosphate uridyltransferase (EC 2.7.7.12) yielded UDP- α-D-galactosamine. The equilibrium of the synthesis was forced to the product side by the addition of phosphoglucomutase (EC 2.7.5.1) and glucose- 6-phosphate dehydrogenase (EC 1.1.1.49). Pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) were used to regenerate UTP and the cofactor NAD. The yield for the enzymatic step was 42%. Finally, UDP-α-D- galactosamine was acetylated chemically with N-acetoxysuccinimide. Product isolation was accomplished by anion-exchange chromatography and gel filtration. The overall yield was 34% and 82 mg UDP-N-acetyl-α-D- galactosamine were isolated.
- Buelter, Thomas,Wandrey, Christian,Elling, Lothar
-
p. 469 - 473
(2007/10/03)
-