CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China.
Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou, China.
Appl Environ Microbiol. 2022 Jan 25;88(2):e0222621. doi: 10.1128/AEM.02226-21. Epub 2021 Nov 24.
Uronic acids are commonly found in marine polysaccharides and increase structural complexity and intrinsic recalcitrance to enzymatic attack. Glycoside hydrolase family 2 (GH2) includes proteins that target sugar conjugates with hexuronates and are involved in the catabolism and cycling of marine polysaccharides. Here, we report a novel GH2, GalA from a marine alga-associated organism with broad substrate specificity. Biochemical analyses revealed that GalA exhibits hydrolyzing activities against β-galacturonide, β-glucuronide, and β-galactopyranoside via retaining mechanisms. We solved the GalA crystal structure in complex with galacturonic acid (GalA) and determined (via mutagenesis) that charge characteristics at uronate-binding subsites controlled substrate selectivity for uronide hydrolysis. Additionally, conformational flexibility of the GalA active-site pocket was proposed as a key component for broad substrate enzyme selectivity. Our GalA structural and functional data augment the current understanding of substrate recognition of GH2 enzymes and provide key insights into the bacterial use of uronic acid-containing polysaccharides. The decomposition of algal glycans driven by marine bacterial communities represents one of the largest heterotrophic transformations of organic matter fueling marine food webs and global carbon cycling. However, our knowledge on carbohydrate cycling is limited due to structural complexity of marine polysaccharides and the complicated enzymatic machinery of marine microbes. To degrade algal glycan, marine bacteria such as members of the produce a complex repertoire of carbohydrate-active enzymes (CAZymes) matching the structural specificities of the different carbohydrates. In this study, we investigated an extracellular GH2 β-glycosidase, GalA from a marine organism, to identify the key components responsible for glycuronide recognition and hydrolysis. The broad substrate specificity of GalA against glycosides with diverse stereochemical substitutions indicates its potential in processing complex marine polysaccharides. Our findings promote a better understanding of microbially driven mechanisms of marine carbohydrate cycling.
糖醛酸通常存在于海洋多糖中，增加了结构的复杂性和对酶攻击的固有抗性。糖苷水解酶家族 2（GH2）包括靶向带有己糖醛酸盐的糖缀合物的蛋白质，并且参与海洋多糖的分解代谢和循环。在这里，我们报告了一种新型 GH2，来自与海洋藻类相关的生物体的 GalA，具有广泛的底物特异性。生化分析表明，GalA 通过保留机制表现出对β-半乳糖苷、β-葡糖苷和β-半乳糖吡喃糖苷的水解活性。我们解决了 GalA 与半乳糖醛酸（GalA）复合物的晶体结构，并通过突变确定了在糖醛酸结合亚基位点的电荷特性控制了对糖醛酸水解的底物选择性。此外，GalA 活性位点口袋的构象灵活性被提出是广泛的底物酶选择性的关键组成部分。我们的 GalA 结构和功能数据增加了对 GH2 酶底物识别的现有理解，并为细菌利用含糖醛酸的多糖提供了关键见解。 由海洋细菌群落驱动的藻类聚糖分解是最大的异养转化之一，为海洋食物网和全球碳循环提供了有机物质燃料。然而，由于海洋多糖的结构复杂性和海洋微生物复杂的酶机制，我们对碳水化合物循环的了解有限。为了降解藻类聚糖，海洋细菌，如海洋生物体的成员，产生了一系列复杂的碳水化合物活性酶（CAZymes），与不同碳水化合物的结构特异性相匹配。在这项研究中，我们研究了一种细胞外 GH2 β-糖苷酶，来自海洋生物体的 GalA，以确定负责糖醛酸识别和水解的关键组成部分。GalA 对具有不同立体化学取代的糖苷的广泛底物特异性表明其在处理复杂海洋多糖方面的潜力。我们的发现促进了对微生物驱动的海洋碳水化合物循环机制的更好理解。
