Abstract
The aerobic soil bacterium Cellvibrio vulgaris has a β-mannan-degradation gene cluster, including unkA, epiA, man5A, and aga27A. Among these genes, epiA has been assigned to encode an epimerase for converting d-mannose to d-glucose, even though the amino acid sequence of EpiA is similar to that of cellobiose 2-epimerases (CEs). UnkA, whose function currently remains unknown, shows a high sequence identity to 4-O-β-d-mannosyl-d-glucose phosphorylase. In this study, we have investigated CE activity of EpiA and the general characteristics of UnkA using recombinant proteins from Escherichia coli. Recombinant EpiA catalyzed the epimerization of the 2-OH group of sugar residue at the reducing end of cellobiose, lactose, and β-(1→4)-mannobiose in a similar manner to other CEs. Furthermore, the reaction efficiency of EpiA for β-(1→4)-mannobiose was 5.5 × 104-fold higher than it was for d-mannose. Recombinant UnkA phosphorolyzed β-d-mannosyl-(1→4)-d-glucose and specifically utilized d-glucose as an acceptor in the reverse reaction, which indicated that UnkA is a typical 4-O-β-d-mannosyl-d-glucose phosphorylase.
摘要:有氧土壤细菌Cellvibrio vulgaris具有一个β-
甘露聚糖降解
基因簇,包括unkA、epiA、man5A和aga27A。在这些
基因中,epiA被指定为编码一个使d-
甘露糖转化为d-
葡萄糖的异构酶,尽管EpiA的
氨基酸序列与赤霉糖2-异构酶(CEs)的相似。UnkA的功能目前仍未知,显示出与4-O-β-d-甘露基-d-
葡萄糖磷酸化酶具有高度相似的序列。在这项研究中,我们使用大肠杆菌的
重组蛋白研究了EpiA的CE活性和UnkA的一般特性。
重组EpiA催化了赤霉糖、
乳糖和β-(1→4)-
甘露二糖末端的糖残基的2-OH基的异构化,类似于其他CEs。此外,EpiA对β-(1→4)-
甘露二糖的反应效率比对d-甘露高5.5×104倍。
重组UnkA对β-d-甘露基-(1→4)-d-
葡萄糖进行了
磷酸解除作用,并在反向反应中特异地利用d-
葡萄糖作为受体,这表明UnkA是一种典型的4-O-β-d-甘露基-d-
葡萄糖磷酸化酶。