Tuning the production of variable length, fluorescent polyisoprenoids using surfactant-controlled enzymatic synthesis.
Tuning the production of variable length, fluorescent polyisoprenoids using surfactant-controlled enzymatic synthesis.
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使用表面活性剂控制的酶合成来调节可变长度、荧光聚异戊二烯的生产。
DOI：
10.1021/acs.biochem.5b00310
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发表时间：
2015
期刊：
影响因子：
2.9
通讯作者：
Dodbele,Samantha
中科院分区：
文献类型：
--
作者：
Troutman,JerryM;Erickson,KatelynM;Scott,PhillipM;Hazel,JosephM;Martinez,ChristinaD;Dodbele,Samantha
二磷酸异戊二烯（BPP）是一种2-Eeight-Z构型的C55类异戊二烯，在细菌的生存和致病过程中起着重要的锚作用。BPP是由聚合酶十一异戊二烯焦磷酸合酶（UppS）催化的，其催化从八个异戊烯基二磷酸延伸具有八个Z-构型异戊二烯单元的单个法呢基二磷酸（FPP）。在这里，使用荧光2-nitrileanilinogeranyl二磷酸类似物的FPP，我们已经发现，广泛的表面活性剂可以刺激释放的产品从UppS和表面活性剂的结构有一个主要的影响，由蛋白质产生的产品的长度。特别重要的是，较短链的表面活性剂促进释放的类异戊二烯与4至sixZ-构型异戊二烯添加，而较大的链表面活性剂促进天然类异戊二烯长度（8 Z）和更大的形成。我们已经发现，产品链长可以很容易地控制和粗调通过调节表面活性剂的身份，浓度和反应时间。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。发现的表面活性剂的影响似乎没有显着改变与替代类异戊二烯基板。然而，表面活性剂的影响似乎是依赖于细菌物种之间的UppS的差异。这项工作提供了新的见解，表面活性剂在酶学的影响，并强调如何利用这些影响，否则难以获得聚糖生物合成探针的化学酶合成。本工作也为系统分析多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。
Bactoprenyl diphosphate (BPP), a two-Eeight-Zconfiguration C55isoprenoid, 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 eightZ-configuration isoprene units from eight isopentenyl diphosphates.In vitroanalysis 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 sixZ-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.
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影响因子：
4.8
作者：
Chang, SY;Ko, TP;Wang, AHJ
通讯作者：
Wang, AHJ
影响因子：
4.8
作者：
T. Touzé;D. Blanot;D. Mengin-Lecreulx
通讯作者：
T. Touzé;D. Blanot;D. Mengin-Lecreulx
影响因子：
2.9
作者：
Chen, L;Men, H;Walker, S
通讯作者：
Walker, S
用于评估细菌酶脂质底物特异性的综合聚异戊二烯醇文库的合成。
影响因子：
2.8
作者：
Wu,Baolin;Woodward,Robert;Wen,Liuqing;Wang,Xuan;Zhao,Guohui;Wang,PengGeorge
通讯作者：
Wang,PengGeorge
影响因子：
64.8
作者：
Ling LL;Schneider T;Peoples AJ;Spoering AL;Engels I;Conlon BP;Mueller A;Schäberle TF;Hughes DE;Epstein S;Jones M;Lazarides L;Steadman VA;Cohen DR;Felix CR;Fetterman KA;Millett WP;Nitti AG;Zullo AM;Chen C;Lewis K
通讯作者：
Lewis K