Active-Site Engineering Switches Carbohydrate Regiospecificity in a Fungal Copper Radical Oxidase,ACS Catalysis

当前位置： X-MOL 学术 › ACS Catal. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Active-Site Engineering Switches Carbohydrate Regiospecificity in a Fungal Copper Radical Oxidase
ACS Catalysis ( IF 11.3 ) Pub Date : 2022-08-05 , DOI: 10.1021/acscatal.2c01956
Yann Mathieu _{1,

2} , Maria E Cleveland _{1,

2,

3} , Harry Brumer _{1,

2,

3,

4,

5}

Affiliation

Copper radical oxidases (CROs) from Auxiliary Activity Family 5, Subfamily 2 (AA5_2), are organic cofactor-free biocatalysts for the selective oxidation of alcohols to the corresponding aldehydes. AA5_2 CROs comprise canonical galactose-6-oxidases as well as the more recently discovered general alcohol oxidases and aryl alcohol oxidases. Guided by primary and tertiary protein structural analyses, we targeted a distinct extended loop in the active site of a Colletotrichum graminicola aryl alcohol oxidase (CgrAAO) to explore its effect on catalysis in the broader context of AA5_2. Deletion of this loop, which is bracketed by a conserved disulfide bridge, significantly reduced the inherent activity of the enzyme toward extended galacto-oligosaccharides, as anticipated from molecular modeling. Unexpectedly, kinetic and product analysis on a range of monosaccharides and disaccharides revealed that an altered carbohydrate specificity in CgrAAO-Δloop was accompanied by a complete change in regiospecificity from C-6 to C-1 oxidation, thereby generating aldonic acids. C-1 regiospecificity is unprecedented in AA5 enzymes and is classically associated with flavin-dependent carbohydrate oxidases of Auxiliary Activity Family 3. Thus, this work further highlights the catalytic adaptability of the unique mononuclear copper radical active site and provides a basis for the design of improved biocatalysts for diverse potential applications.

中文翻译：

活性位点工程切换真菌铜自由基氧化酶中的碳水化合物区域特异性

来自辅助活性家族 5 亚家族 2 (AA5_2) 的铜自由基氧化酶 (CRO) 是有机无辅因子生物催化剂，用于将醇选择性氧化为相应的醛。 AA5_2 CRO 包含典型的半乳糖-6-氧化酶以及最近发现的一般醇氧化酶和芳基醇氧化酶。在一级和三级蛋白质结构分析的指导下，我们针对炭疽病菌芳醇氧化酶 ( Cgr AAO) 活性位点中的一个独特的延伸环，探索其在 AA5_2 更广泛的背景下对催化的影响。正如分子模型所预期的那样，删除由保守二硫桥包围的这个环，显着降低了酶对延长的低聚半乳糖的固有活性。出乎意料的是，对一系列单糖和二糖的动力学和产物分析表明， Cgr AAO-Δloop 中碳水化合物特异性的改变伴随着区域特异性从 C-6 氧化到 C-1 氧化的完全变化，从而产生醛糖酸。 C-1区域特异性在AA5酶中是前所未有的，并且通常与辅助活性家族3的黄素依赖性碳水化合物氧化酶相关。因此，这项工作进一步强调了独特的单核铜自由基活性位点的催化适应性，并为设计提供了基础改进的生物催化剂适用于多种潜在应用。

更新日期：2022-08-05

点击分享查看原文

点击收藏

阅读更多本刊最新论文本刊介绍/投稿指南11