Reconstitution and optimisation of the biosynthesis of bacterial sugar pseudaminic acid (Pse5Ac7Ac) enables preparative enzymatic synthesis of CMP-Pse5Ac7Ac.
Reconstitution and optimisation of the biosynthesis of bacterial sugar pseudaminic acid (Pse5Ac7Ac) enables preparative enzymatic synthesis of CMP-Pse5Ac7Ac.
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DOI：
10.1038/s41598-021-83707-x
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发表时间：
2021-02-26
影响因子：
4.6
通讯作者：
Fascione MA
中科院分区：
文献类型：
--
作者：
Chidwick HS;Flack EKP;Keenan T;Walton J;Thomas GH;Fascione MA
存在于致病菌（包括肠道病原体空肠弯曲杆菌和幽门螺杆菌）表面上的假氨基酸被假定通过调节鞭毛组装和人类免疫系统对细菌的识别而在相关感染性疾病的病因学中发挥有影响的作用。然而，与糖科学的其他领域相比，它们的探索不足，特别是负责细菌中这些糖的糖基转移的酶仍然没有明确的特征。这可以在很大程度上归因于缺乏获得核苷酸活化的假氨基酸糖基供体，如CMP-Pse 5Ac 7Ac。在此，我们使用来自C.空肠弯曲菌（PseBCHGI）在该过程中使用氘洗涤优化与PseBC的偶联周转，并建立PseH tunover中辅因子再生的方法。此外，我们建立了从嗜水气单胞菌中纯化可溶性CMP-Pse 5Ac 7Ac合成酶PseF的条件，并将其与C.空肠酶实现CMP-Pse 5Ac 7Ac的体外实用制备合成，促进未来的生物学研究。
Pseudaminic acids present on the surface of pathogenic bacteria, including gut pathogens Campylobacter jejuni and Helicobacter pylori, are postulated to play influential roles in the etiology of associated infectious diseases through modulating flagella assembly and recognition of bacteria by the human immune system. Yet they are underexplored compared to other areas of glycoscience, in particular enzymes responsible for the glycosyltransfer of these sugars in bacteria are still to be unambiguously characterised. This can be largely attributed to a lack of access to nucleotide-activated pseudaminic acid glycosyl donors, such as CMP-Pse5Ac7Ac. Herein we reconstitute the biosynthesis of Pse5Ac7Ac in vitro using enzymes from C. jejuni (PseBCHGI) in the process optimising coupled turnover with PseBC using deuterium wash in experiments, and establishing a method for co-factor regeneration in PseH tunover. Furthermore we establish conditions for purification of a soluble CMP-Pse5Ac7Ac synthetase enzyme PseF from Aeromonas caviae and utilise it in combination with the C. jejuni enzymes to achieve practical preparative synthesis of CMP-Pse5Ac7Ac in vitro, facilitating future biological studies.
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影响因子：
3.6
作者：
Schirm, M;Soo, EC;Logan, SM
通讯作者：
Logan, SM
影响因子：
3.6
作者：
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通讯作者：
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3.2
作者：
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通讯作者：
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影响因子：
3.1
作者：
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通讯作者：
Knirel, Yuriy A.
影响因子：
4.8
作者：
Schoenhofen, IC;Lunin, VV;Young, NM
通讯作者：
Young, NM