- Substrate specificity of the macrolide-glycosylating enzyme pair DesVII/DesVIII: Opportunities, limitations, and mechanistic hypotheses
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(Chemical Equation Presented) Two's Company: DesVII, a glycosyltransferase involved in the biosynthesis of macrolide antibiotics, is unusual in that it requires an additional protein partner, DesVIII, for its full activity. The level of substrate tolerance of the DesVII/DesVIII pair was explored.
- Borisova, Svetlana A.,Zhang, Changsheng,Takahashi, Haruko,Zhang, Hua,Wong, Alexander W.,Thorson, Jon S.,Liu, Hung-Wen
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- Target-specific identification and characterization of the putative gene cluster for brasilinolide biosynthesis revealing the mechanistic insights and combinatorial synthetic utility of 2-deoxy-l-fucose biosynthetic enzymes
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Brasilinolides exhibiting potent immunosuppressive and antifungal activities with remarkably low toxicity are structurally characterized by an unusual modified 2-deoxy-l-fucose (2dF) attached to a type I polyketide (PK-I) macrolactone. From the pathogenic producer Nocardia terpenica (Nocardia brasiliensis IFM-0406), a 210 kb genomic fragment was identified by target-specific degenerate primers and subsequently sequenced, revealing a giant nbr gene cluster harboring genes (nbrCDEF) required for TDP-2dF biosynthesis and those for PK-I biosynthesis, modification and regulation. The results showed that the genetic and domain arrangements of nbr PK-I synthases agreed colinearly with the PK-I structures of brasilinolides. Subsequent heterologous expression of nbrCDEF in Escherichia coli accomplished in vitro reconstitution of TDP-2dF biosynthesis. The catalytic functions and mechanisms of NbrCDEF enzymes were further characterized by systematic mix-and-match experiments. The enzymes were revealed to display remarkable substrate and partner promiscuity, leading to the establishment of in vitro hybrid deoxysugar biosynthetic pathways throughout an in situ one-pot (iSOP) method. This study represents the first demonstration of TDP-2dF biosynthesis at the enzyme and molecular levels, and provides new hope for expanding the structural diversity of brasilinolides by combinatorial biosynthesis.
- Chiu, Hsien-Tai,Weng, Chien-Pao,Lin, Yu-Chin,Chen, Kuan-Hung
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supporting information
p. 1988 - 2006
(2016/02/18)
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- One-pot four-enzyme synthesis of thymidinediphosphate-l-rhamnose
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A new, robust one-pot four-enzyme synthetic method was developed for thymidinediphosphate-l-rhamnose starting from d-glucose-1-phosphate. The enzymes, Glc-1-P thymidylyltransferase, dTDP-Glc-4,6-dehydratase, dTDP-4-keto-6-deoxy-Glc-3,5-epimerase and dTDP-4-keto-Rha reductase were derived from Streptococcus pneumonia serotype 23F, expressed in Escherichia coli, and studied in detail to provide the first direct evidence for their functions.
- Li, Siqiang,Wang, Hong,Ma, Juncai,Gu, Guofeng,Chen, Zonggang,Guo, Zhongwu
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p. 13995 - 13998
(2016/12/09)
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- Mechanistic studies of the radical S-adenosylmethionine enzyme DesII with TDP-D-fucose
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DesII is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the C4-deamination of TDP-4-amino-4,6-dideoxyglucose through a C3 radical intermediate. However, if the C4 amino group is replaced with a hydroxy group (to give TDP-quinovose), the hydroxy group at C3 is oxidized to a ketone with no C4-dehydration. It is hypothesized that hyperconjugation between the C4 C-N/O bond and the partially filled p orbital at C3 of the radical intermediate modulates the degree to which elimination competes with dehydrogenation. To investigate this hypothesis, the reaction of DesII with the C4-epimer of TDP-quinovose (TDP-fucose) was examined. The reaction primarily results in the formation of TDP-6-deoxygulose and likely regeneration of TDP-fucose. The remainder of the substrate radical partitions roughly equally between C3-dehydrogenation and C4-dehydration. Thus, changing the stereochemistry at C4 permits a more balanced competition between elimination and dehydrogenation.
- Ko, Yeonjin,Ruszczycky, Mark W.,Choi, Sei-Hyun,Liu, Hung-Wen
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p. 860 - 863
(2015/03/05)
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- dTDP-BETA-D-FUCOFURANOSE, ITS PREPARATION METHOD AND USE
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Provided is a dTDP-β-D-fucofuranose, which is also referred to as dTDP-β-6-deoxy-D-galactofuranose. The dTDP-β-D-fucofuranose is synthesized by using reductase Fcf1 and mutase Fcf2 in the gram-negative bacteria. Also provided are the preparation method of the dTDP-β-D-fucofuranose and use of the dTDP-β-D-fucofuranose for manufacturing a medicament for the treatment of tumors.
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Page/Page column 4
(2012/03/08)
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- Exploiting nucleotidylyltransferases to prepare sugar nucleotides
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(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.
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p. 857 - 860
(2007/10/03)
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- One-step synthesis of labeled sugar nucleotides for protein O-GlcNAc modification studies by chemical function analysis of an archaeal protein
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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.
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p. 836 - 837
(2007/10/03)
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- Unusually broad substrate tolerance of a heat-stable archaeal sugar nucleotidyltransferase for the synthesis of sugar nucleotides
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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.
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p. 15993 - 15998
(2007/10/03)
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- Active-site engineering of nucleotidylyltransferases and general enzymatic methods for the synthesis of natural and "unnatural" UDP- and TDP-nucleotide sugars
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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.
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- A novel method for the preparation of nucleoside diphosphates
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Figure presented Sugar nucleoside diphosphates have been prepared using an efficient phosphate coupling reaction that employs a highly reactive zwitterionic phosphoramidate intermediate as the phosphorylating species.
- Freel Meyers, Caren L.,Borch, Richard F.
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p. 3765 - 3768
(2007/10/03)
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- Synthesis of homorhamnojirimycins and related trihydroxypipecolic acid derivatives via divergent bicyclic amino lactone intermediates: Inhibition of naringinase (L-rhamnosidase) and dTDP-rhamnose biosynthesis
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A series of homorhamnojirimycins and related compounds are prepared from two epimeric [2.2.2] bicyclic amino lactones 6 and 7 via the 2-azidoheptono-1,5-lactone 8, itself derived from L-rhamnose. Aminolysis and deprotection of the bicyclic lactones provid
- Shilvock, John P.,Wheatley, Joseph R.,Nash, Robert J.,Watson, Alison A.,Griffiths, Rhodri C.,Butters, Terry D.,Mueller, Mathias,Watkin, David J.,Winkler, David A.,Fleet, George W. J.
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p. 2735 - 2746
(2007/10/03)
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