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Determinants of the Nucleotide Specificity in the Carbohydrate Epimerase Family 1.
Biotechnology Journal ( IF 3.2 ) Pub Date : 2020-08-06 , DOI: 10.1002/biot.202000132
Stevie Van Overtveldt 1 , Matthieu Da Costa 1 , Ophelia Gevaert 1 , Henk-Jan Joosten 2 , Koen Beerens 1 , Tom Desmet 1
Affiliation  

In recent years, carbohydrate epimerases have attracted increasing attention as promising biocatalysts for the production of specialty sugars and derivatives. The vast majority of these enzymes are active on nucleotide‐activated sugars, rather than on their free counterparts. Although such epimerases are known to have a clear preference for a particular nucleotide (UDP, GDP, CDP, or ADP), very little is known about the determinants of the respective specificities. In this work, sequence motifs are identified that correlate with the different nucleotide specificities in one of the main epimerase superfamilies, carbohydrate epimerase 1 (CEP1). To confirm their relevance, GDP‐ and CDP‐specific residues are introduced into the UDP‐glucose 4‐epimerase from Thermus thermophilus, resulting in a 3‐fold and 13‐fold reduction in KM for GDP‐Glc and CDP‐Glc, respectively. Moreover, several variants are severely crippled in UDP‐Glc activity, which further underlines the crucial role of the identified positions. Hence, the analysis should prove to be valuable for the further exploration and application of epimerases involved in carbohydrate synthesis.

中文翻译:

糖差向异构酶家族中核苷酸特异性的决定因素1。

近年来,碳水化合物差向异构酶作为生产特种糖及其衍生物的有前途的生物催化剂已引起越来越多的关注。这些酶中的绝大多数对核苷酸激活的糖具有活性,而不是其游离的同种酶。尽管已知此类差向异构酶对特定核苷酸(UDP,GDP,CDP或ADP)具有明显的偏好,但对各个特异性的决定因素了解甚少。在这项工作中,确定了与主要差向异构酶超家族之一,碳水化合物差向异构酶1(CEP1)中不同核苷酸特异性相关的序列基序。为了确认它们的相关性,将特定于GDP和CDP的残基引入嗜热栖热菌的UDP-葡萄糖4-表异构酶,导致GDP‐Glc和CDP‐Glc的K M分别降低3倍和13倍。此外,UDP-Glc活动严重破坏了多个变体,这进一步突显了所确定位置的关键作用。因此,该分析应被证明对于进一步探索和应用与碳水化合物合成有关的差向异构酶是有价值的。
更新日期:2020-08-06
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