Characterization of a thermotolerant ROK-type mannofructokinase from Streptococcus mitis: application to the synthesis of phosphorylated sugars
作者:Carine Vergne-Vaxelaire、Aline Mariage、Jean-Louis Petit、Aurélie Fossey-Jouenne、Christine Guérard-Hélaine、Ekaterina Darii、Adrien Debard、Stessy Nepert、Virginie Pellouin、Marielle Lemaire、Anne Zaparucha、Marcel Salanoubat、Véronique de Berardinis
DOI:10.1007/s00253-018-9018-1
日期:2018.7
As proof of concept, 8 hexoses were phosphorylated in moderate to good yields, some of them described for the first time like L-sorbose-5-phosphate unusually phosphorylated in position 5. Its thermotolerance, its wide pH tolerance (from 7 to 10), and temperature range (> 85% activity between 40 and 70 °C) open the way to applications in the enzymatic synthesis of monophosphorylated hexoses.
迄今为止,大多数已经表征且表现出激酶活性的“阻遏物,开放阅读框,激酶”(ROK)蛋白都以D-葡萄糖为底物。通过探索测序的细菌多样性,已鉴定出61个属于ROK家族的ATP依赖性激酶,并通过实验测定了其己糖的磷酸化程度。主要发现这些激酶是耐热的,并且对D-甘露糖和D-果糖具有高活性,对D-塔格糖具有显着的活性。其中,对中嗜性链球菌的炎症(称为ScrKmitis)的ATP依赖性激酶进行了生物化学表征,并对其底物谱进行了进一步研究。该酶对D-甘露糖和D-果糖的催化效率分别为1.5 106 s-1 M-1和2.7 105 s-1 M-1。但对D-塔格糖(3.5 102 s-1 M-1)和非天然单糖D-altrose(1.1 104 s-1 M-1)和D-塔洛糖(3.4 102 s-1 M-1)也是重要的。对所有己糖测得的比活度显示出对D系列优于L系列的立体偏好。作为概念证明,8己糖以中等到良好的产率