Chemoenzymatic synthesis of CMP-sialic acid derivatives by a one-pot two-enzyme system: comparison of substrate flexibility of three microbial CMP-sialic acid synthetases
Chemoenzymatic synthesis of CMP-sialic acid derivatives by a one-pot two-enzyme system: comparison of substrate flexibility of three microbial CMP-sialic acid synthetases
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DOI：
10.1016/j.bmc.2004.09.030
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发表时间：
2004-12-15
影响因子：
3.5
通讯作者：
Chen, X
中科院分区：
文献类型：
--
作者：
Yu, H;Yu, H;Chen, X
分别从脑膜炎奈瑟菌B群、无乳链球菌血清型V和大肠杆菌K1中克隆的三种C末端His（6）-标记的重组微生物CMP-唾液酸合成酶[EC 2.7.7.43]评价它们在一锅双酶系统中合成CMP-唾液酸衍生物的能力。在该系统中，N-乙酰甘露糖胺或甘露糖类似物与丙酮酸缩合，由从E. 4.1.3.3 coliK 12，以提供唾液酸类似物作为CMP-唾液酸合成酶的底物。底物的灵活性和反应效率的三个重组CMP-唾液酸合成酶进行了比较，首先通过使用薄层色谱定性筛选，然后通过使用高效液相色谱定量分析。了N.在三种合成酶中，脑膜炎合成酶的表达水平最高，底物特异性最灵活，催化效率最高。最后，以制备规模（100-200 mg）从它们的5-或6-碳糖前体使用N.脑膜炎合成酶和醛缩酶。(C)2004爱思唯尔有限公司保留所有权利。
Three C terminal His(6)-tagged recombinant microbial CMP-sialic acid synthetases [EC 2.7.7.43] cloned from Neisseria meningitidis group B, Streptococcus agalactiae serotype V, and Escherichia coli K1, respectively, were evaluated for their ability in the synthesis of CMP-sialic acid derivatives in a one-pot two-enzyme system. In this system, N-acetylmannosamine or mannose analogs were condensed with pyruvate, catalyzed by a recombinant sialic acid aldolase [EC 4.1.3.3] cloned from E. coli K12 to provide sialic acid analogs as substrates for the CMP-sialic acid synthetases. The substrate flexibility and the reaction efficiency of the three recombinant CMP-sialic acid synthetases were compared, first by qualitative screening using thin layer chromatography, and then by quantitative analysis using high performance liquid chromatography. The N. meningitidis synthetase was shown to have the highest expression level, the most flexible substrate specificity, and the highest catalytic efficiency among the three synthetases. Finally, eight sugar nucleotides, including cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) and its derivatives with substitutions at carbon-5, carbon-8, or carbon-9 of Neu5Ac, were synthesized in a preparative (100-200 mg) scale from their 5- or 6-carbon sugar precursors using the N. meningitidis synthetase and the aldolase. (C) 2004 Elsevier Ltd. All rights reserved.