Effective Synthesis of Guanosine 5′‐Diphospho‐β‐l‐galactose Using Bacterial l‐Fucokinase/Guanosine 5′‐Diphosphate‐l‐fucose Pyrophosphorylase,Advanced Synthesis & Catalysis

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Effective Synthesis of Guanosine 5′‐Diphospho‐β‐l‐galactose Using Bacterial l‐Fucokinase/Guanosine 5′‐Diphosphate‐l‐fucose Pyrophosphorylase
Advanced Synthesis & Catalysis ( IF 4.4 ) Pub Date : 2017-11-30 , DOI: 10.1002/adsc.201700901
Hiroyuki Ohashi ₁ , Claudia Wahl ₂ , Takao Ohashi ₁ , Lothar Elling ₂ , Kazuhito Fujiyama ₁

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The nucleotide sugar guanosine 5′‐diphospho‐β‐l‐galactose (GDP‐l‐Gal) is known as a key intermediate of the l‐ascorbic acid biosynthesis pathway of plants and algae. In addition, GDP‐l‐Gal serves as a donor substrate of l‐galactosyltransferase, which transfers l‐Gal on the non‐reducing ends of glycoconjugates. To synthesize varieties of l‐Gal‐containing glycoconjugates and explore the novel l‐galactosyltransferases, GDP‐l‐Gal needs to be prepared since it is not commercially available. In plants, GDP‐d‐mannose‐3′,5′‐epimerase (GME) converts GDP‐α‐d‐mannose (GDP‐d‐Man) to GDP‐l‐Gal and GDP‐l‐gulose. GDP‐l‐Gal has been previously prepared using GME and GDP‐d‐Man, for which the equilibrium ratio was biased to GDP‐d‐Man. In this study, the efficient GDP‐l‐Gal production from l‐Gal was established using bacterial l‐fucokinase/GDP‐l‐fucose pyrophosphorylase (FKP), l‐fucose (l‐Fuc), adenosine 5′‐triphosphate (ATP) and guanosine 5′‐triphosphate (GTP). A high synthesis yield was obtained after screening of the optimum FKP reaction conditions in a 96‐well format and analysis by multiplexed capillary electrophoresis (MP‐CE). Conversion of l‐Gal substrate yielded 97% GDP‐l‐Gal using purified recombinant FKP expressed in E. coli. Moreover, GDP‐l‐Gal was successfully purified with 92% (34.5 mg) overall yield from the FKP reaction mixture. GDP‐l‐Gal is now readily available for studies on l‐galactosyltransferases and l‐fucosyltransferases.

中文翻译：

使用细菌l-岩藻糖激酶/番石榴5'-二磷酸l-岩藻糖焦磷酸化酶有效合成鸟苷5'-二磷酸-β-l-半乳糖

核苷酸糖鸟苷5'-二磷酸-β-升半乳糖（GDP-升-Gal）被称为所述的关键中间体升植物和藻类的抗坏血酸生物合成途径。此外，GDP-升-Gal充当的施主衬底升-galactosyltransferase，其转让升-Gal上的糖缀合物的非还原末端。为了合成品种升含-Gal-糖缀合物和探索新的升-galactosyltransferases，GDP-升待制备-Gal需求，因为它不是可商购的。在植物中，GDP- d-甘露糖-3'，5'-表异构酶（GME）转换GDP-α- d-甘露糖（GDP- d- Man ）到GDP- l- Gal和GDP- l- gulose。GDPl - Gal以前是使用GME和GDP- d- Man来准备的，因此均衡比率相对于GDP- d- Man有偏差。在这项研究中，有效的GDP-升从-Gal生产升-Gal使用细菌建立升-fucokinase / GDP-升岩藻糖焦磷酸化酶（FKP），升-岩藻糖（升-Fuc），5'-三磷酸腺苷（ATP）和5'-三磷酸鸟苷（GTP）。在以96孔格式筛选最佳FKP反应条件并通过多重毛细管电泳（MP-CE）分析后，获得了很高的合成产率。使用在大肠杆菌中表达的纯化重组FKP，对l- Gal底物的转化产生了97％的GDP- l- Gal 。此外，从FKP反应混合物中成功纯化了GDPl - Gal，总产率为92％（34.5 mg）。GDP- l- Gal现在可用于研究l-半乳糖基转移酶和l-岩藻糖基转移酶。

更新日期：2017-11-30

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