Troutman Jerry M, Erickson Katelyn M, Scott Phillip M, Hazel Joseph M, Martinez Christina D, Dodbele Samantha
†Department of Chemistry, ‡Nanoscale Science Program, and §The Center for Biomedical Engineering and Science, University of North Carolina at Charlotte, 9201 University City Boulevard, Charlotte, North Carolina 28223, United States.
Biochemistry. 2015 May 12;54(18):2817-27. doi: 10.1021/acs.biochem.5b00310. Epub 2015 Apr 29.
Bactoprenyl diphosphate (BPP), a two-E eight-Z configuration C55 isoprenoid, serves as a critical anchor for the biosynthesis of complex glycans central to bacterial survival and pathogenesis. BPP is formed by the polymerase undecaprenyl pyrophosphate synthase (UppS), which catalyzes the elongation of a single farnesyl diphosphate (FPP) with eight Z-configuration isoprene units from eight isopentenyl diphosphates. In vitro analysis of UppS and other polyprenyl diphosphate synthases requires the addition of a surfactant such as Triton X-100 to stimulate the release of the hydrophobic product from the enzyme for effective and efficient turnover. Here using a fluorescent 2-nitrileanilinogeranyl diphosphate analogue of FPP, we have found that a wide range of surfactants can stimulate release of product from UppS and that the structure of the surfactant has a major impact on the lengths of products produced by the protein. Of particular importance, shorter chain surfactants promote the release of isoprenoids with four to six Z-configuration isoprene additions, while larger chain surfactants promote the formation of natural isoprenoid lengths (8Z) and larger. We have found that the product chain lengths can be readily controlled and coarsely tuned by adjusting surfactant identity, concentration, and reaction time. We have also found that binary mixtures of just two surfactants can be used to fine-tune isoprenoid lengths. The surfactant effects discovered do not appear to be significantly altered with an alternative isoprenoid substrate. However, the surfactant effects do appear to be dependent on differences in UppS between bacterial species. This work provides new insights into surfactant effects in enzymology and highlights how these effects can be leveraged for the chemoenzymatic synthesis of otherwise difficult to obtain glycan biosynthesis probes. This work also provides key reagents for the systematic analysis of structure-activity relationships between glycan biosynthesis enzymes and isoprenoid structure.
细菌萜醇二磷酸（BPP）是一种具有两个反式双键、八个顺式双键构型的C55类异戊二烯，是细菌生存和致病所必需的复杂聚糖生物合成的关键锚定物。BPP由聚合酶十一异戊烯焦磷酸合酶（UppS）形成，该酶催化一个法尼基二磷酸（FPP）与八个来自异戊烯基二磷酸的顺式双键构型异戊二烯单元延长。对UppS和其他聚异戊二烯二磷酸合酶的体外分析需要添加一种表面活性剂，如 Triton X-100，以刺激疏水产物从酶中释放出来，从而实现有效且高效的周转。在这里，我们使用FPP的荧光2-硝基苯胺香叶基二磷酸类似物，发现多种表面活性剂可以刺激UppS释放产物，并且表面活性剂的结构对该蛋白产生的产物长度有重大影响。特别重要的是，短链表面活性剂促进添加了四个至六个顺式双键构型异戊二烯的类异戊二烯的释放，而长链表面活性剂促进天然类异戊二烯长度（8个顺式双键）及更长产物的形成。我们发现，通过调整表面活性剂的种类、浓度和反应时间，可以很容易地控制和粗略调节产物链长度。我们还发现，仅两种表面活性剂的二元混合物可用于微调类异戊二烯长度。发现的表面活性剂效应似乎不会因替代类异戊二烯底物而发生显著改变。然而，表面活性剂效应似乎确实取决于细菌物种之间UppS的差异。这项工作为酶学中的表面活性剂效应提供了新的见解，并突出了如何利用这些效应进行化学酶促合成难以获得的聚糖生物合成探针。这项工作还为系统分析聚糖生物合成酶与类异戊二烯结构之间的构效关系提供了关键试剂。
