- 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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- 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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- 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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