General Utilization of Fluorescent Polyisoprenoids with Sugar Selective Phosphoglycosyltransferases.
General Utilization of Fluorescent Polyisoprenoids with Sugar Selective Phosphoglycosyltransferases.
复制标题
荧光聚异戊二烯类化合物与糖选择性磷酸糖基转移酶的一般用途。
DOI：
10.1021/acs.biochem.9b01026
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发表时间：
2020
期刊：
影响因子：
2.9
通讯作者：
Troutman,JerryM
中科院分区：
文献类型：
--
作者：
Reid,AmandaJ;Scarbrough,BethA;Williams,TiffanyC;Gates,ClaireE;Eade,ColleenR;Troutman,JerryM
细菌的保护性表面由多糖组成，并参与宿主入侵和定殖、宿主免疫系统逃避和抗菌剂抗性。我们对这些复杂的表面多糖的基本理解的主要障碍在于细菌物种之间聚糖组成的巨大多样性。聚类异戊二烯bactoprenyl磷酸（或十一异戊二烯磷酸）是糖聚合物组装早期阶段所必需的基本脂质载体。由于bactoprenyl磷酸在这些关键过程中的普遍存在，附加到这种脂质载体的分子探针简化了多糖生物组装过程中酶作用的鉴定。这些探针的数量有限，存在于文献中或已与这些途径进行了评估，其使用的限制目前尚不清楚。在此，我们设计了一种用于生产荧光修饰的bactoprenyl探针的有效方法。我们进一步扩大了我们以前的努力，利用2-腈苯胺，并首次额外制备硝基苯并恶二唑标记的bactoprenyl磷酸盐。然后，我们评估这两个探针的酶混杂利用四个良好的特点启动磷酸糖基转移酶：CPS 2 E（肺炎链球菌），WbaP（沙门氏菌），WecA（大肠杆菌），和WecP（气单胞菌）。这两种探针作为这些酶的底物，可以很容易地用于研究广泛的细菌糖组装途径。有趣的是，我们还确定了独特的溶解度要求的硝基苯并恶二唑部分有效的酶的利用，没有观察到的2-nitrileaniline。
The protective surfaces of bacteria are comprised of polysaccharides and are involved in host invasion and colonization, host immune system evasion, and antibacterial resistance. A major barrier to our fundamental understanding of these complex surface polysaccharides lies in the tremendous diversity in glycan composition among bacterial species. The polyisoprenoid bactoprenyl phosphate (or undecaprenyl phosphate) is an essential lipid carrier necessary for early stages of glycopolymer assembly. Because of the ubiquity of bactoprenyl phosphate in these critical processes, molecular probes appended to this lipid carrier simplify identification of enzymatic roles during polysaccharide bioassembly. A limited number of these probes exist in the literature or have been assessed with such pathways, and the limits of their use are not currently known. Herein, we devise an efficient method for producing fluorescently modified bactoprenyl probes. We further expand our previous efforts utilizing 2-nitrileaniline and additionally prepare nitrobenzoxadizol-tagged bactoprenyl phosphate for the first time. We then assess the enzyme promiscuity of these two probes utilizing four well-characterized initiating phosphoglycosyltransferases: CPS2E (Streptococcus pneumoniae), WbaP (Salmonella enterica), WecA (Escherichia coli), and WecP (Aeromonas hydrophilia). Both probes serve as substrates for these enzymes and could be readily used to investigate a wide range of bacterial glycoassembly pathways. Interestingly, we have also identified unique solubility requirements for the nitrobenzoxadizol moiety for efficient enzymatic utilization that was not observed for the 2-nitrileaniline.