Investigations on the Determinants Responsible for Low Molar Mass Dextran Formation by DSR-M Dextransucrase,ACS Catalysis

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Investigations on the Determinants Responsible for Low Molar Mass Dextran Formation by DSR-M Dextransucrase
ACS Catalysis ( IF 11.3 ) Pub Date : 2017-09-22 00:00:00 , DOI: 10.1021/acscatal.7b02182
Marion Claverie ₁ , Gianluca Cioci ₁ , Marlène Vuillemin ₁ , Nelly Monties ₁ , Pierre Roblin ₂ , Guy Lippens ₁ , Magali Remaud-Simeon ₁ , Claire Moulis ₁

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Certain enzymes of the GH70 family dextransucrases synthesize very high molar mass dextran polymers, whereas others produce a mixed population of very high and low molar mass products directly from sucrose substrate. Identifying the determinants dictating polymer elongation would allow the tight control of dextran size. To explore this central question, we focus on the recently discovered DSR-M enzyme from Leuconostoc citreum NRRL B-1299, which is the sole enzyme that naturally, exclusively, and very efficiently produces only low molar mass dextrans from sucrose. Extensive biochemical and structural characterization of a truncated form of DSR-M (DSR-MΔ2, displaying the same biochemical behavior as the parental enzyme) and X-ray structural analysis of complexes with sucrose and isomaltotetraose molecules together with accurate monitoring of the resulting polymer formation reveal that DSR-MΔ2 adopts a nonprocessive mechanism attributed to (i) a high propensity to recognize sucrose as a preferred acceptor at the initial stage of catalysis, (ii) an ability to elongate oligodextrans irrespective of their size, and (iii) the presence of a domain V showing a weak ability to bind to the growing dextran chains. In this study, we present the 3D structure with the largest defined domain V reported to date in the GH70 family and map sugar binding pockets on the basis of the structure of the complex obtained with isomaltotetraose. Altogether, these findings give insights into the interplay between the domain V and the catalytic site during polymerization. They open promising strategies for GH70 enzyme engineering aiming at modulating glucan size.

中文翻译：

DSR-M Dextransucrase负责低摩尔质量右旋糖酐形成的决定因素研究

GH70家族葡聚糖的某些酶可合成非常高摩尔质量的右旋糖酐聚合物，而其他酶则直接从蔗糖底物中产生非常高和低摩尔质量的产物的混合群体。确定决定聚合物伸长率的决定因素将允许对葡聚糖尺寸的严格控制。为了探究这个核心问题，我们集中在最近发现的柠檬亮褐藻中的DSR-M酶上。NRRL B-1299，这是唯一的天然，排他且非常有效地从蔗糖中产生低摩尔质量右旋糖酐的酶。截短形式的DSR-M（DSR-MΔ2，表现出与亲本酶相同的生化行为）的广泛生化和结构表征，以及蔗糖和异麦芽四糖分子配合物的X射线结构分析，以及对形成的聚合物形成的精确监控揭示DSR-MΔ2采用了一种非过程性机制，这归因于（i）在催化初期很容易将蔗糖识别为优选的受体，（ii）不论其大小如何都具有延长寡聚葡聚糖的能力，以及（iii）存在域V显示出与增长的葡聚糖链结合的能力较弱。在这项研究中，我们介绍了GH70家族中迄今为止报道的最大定义域V的3D结构，并根据异麦芽四糖复合物的结构图绘制了糖结合口袋。总而言之，这些发现提供了对在聚合过程中结构域V和催化位点之间相互作用的见解。他们为GH70酶工程学打开了有希望的策略，旨在调节葡聚糖的大小。

更新日期：2017-09-22

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