Site-selective CE4 enzyme from Rhodothermus marinus and diversified LmbE-Like catalysis in Bacillus cereus

IF 3.4 3区化学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Carbohydrate Research Pub Date : 2026-02-01 Epub Date: 2025-12-13 DOI:10.1016/j.carres.2025.109792

Jun Bian, Pai Zang, Ran Tang, Can Wei, Yaqing Wang, Li Liu, Josef Voglmeir

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Abstract

Chitooligosaccharides (COS) display diverse biological functions that depend strongly on their N-acetylation pattern, motivating enzymatic approaches that enable regioselective deacetylation. Here, we identified a CE4 deacetylase, RmCBDA2510 from Rhodothermus marinus that performs strict, site-selective, and chain-length-dependent mono-deacetylation of COS. Exoglycosidase probing together with MALDI-TOF/TOF MS/MS showed that RmCBDA2510 precisely generates AD (GlcNAc-GlcN), ADA (GlcNAc-GlcN-GlcNAc), and AADA (GlcNAc-GlcNAc-GlcN-GlcNAc) from chitobiose, chitotriose, and chitotetraose, respectively, revealing a distinctive internal subsite preference. The enzyme is thermophilic and alkaline-active, and site-directed mutagenesis identified Asp41 as essential for catalysis, establishing RmCBDA2510 as a highly selective biocatalyst for producing structurally uniform, partially deacetylated COS.

In contrast, the LmbE-like enzyme BCLmbE1534 from Bacillus cereus catalyzes both the deacetylation of N-acetylglucosamine glycosides and acylation/trans-acylation with short-chain fatty acids and biotin. This represents the first demonstration of trans-acylation within the LmbE family and expands its catalytic repertoire beyond simple deacetylation. Together, RmCBDA2510 and BCLmbE1534 provide complementary enzymatic platforms for tailoring COS structures and diversifying amino-sugar derivatives, thereby broadening the synthetic utility of carbohydrate-active deacetylases.

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marinus Rhodothermus的位点选择性CE4酶及其在蜡样芽孢杆菌中的多种lmbe样催化作用。

壳寡糖（COS）显示出多种生物学功能，这些功能强烈依赖于它们的n -乙酰化模式，从而激发酶的方法来实现区域选择性去乙酰化。在这里，我们从海Rhodothermus marinus中鉴定了一种CE4去乙酰化酶RmCBDA2510，该酶能对COS进行严格的、位点选择性的、链长依赖性的单去乙酰化。外糖苷酶检测结合MALDI-TOF/TOF质谱/质谱分析显示，RmCBDA2510分别从壳聚糖、壳三糖和壳四糖中精确生成AD （GlcNAc-GlcN）、ADA （GlcNAc-GlcN- glcnac）和AADA (GlcNAc-GlcNAc-GlcN-GlcNAc)，显示出独特的内部亚位偏好。该酶嗜热和碱活性，位点定向诱变鉴定出Asp41是催化的必要条件，建立了RmCBDA2510作为一种高度选择性的生物催化剂，用于生产结构均匀，部分去乙酰化的COS。相比之下，蜡样芽孢杆菌的lmbe样酶BCLmbE1534既能催化n -乙酰氨基葡萄糖苷的去乙酰化，也能催化短链脂肪酸和生物素的酰化/反酰化。这代表了LmbE家族中反式酰化的第一次演示，并扩展了其催化功能，超越了简单的去乙酰化。RmCBDA2510和BCLmbE1534共同为裁剪COS结构和多样化氨基糖衍生物提供了互补的酶促平台，从而扩大了碳水化合物活性脱乙酰酶的合成用途。

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来源期刊

Carbohydrate Research 化学-生化与分子生物学

CiteScore

5.00

自引率

3.20%

发文量

183

审稿时长

3.6 weeks

期刊介绍： Carbohydrate Research publishes reports of original research in the following areas of carbohydrate science: action of enzymes, analytical chemistry, biochemistry (biosynthesis, degradation, structural and functional biochemistry, conformation, molecular recognition, enzyme mechanisms, carbohydrate-processing enzymes, including glycosidases and glycosyltransferases), chemical synthesis, isolation of natural products, physicochemical studies, reactions and their mechanisms, the study of structures and stereochemistry, and technological aspects. Papers on polysaccharides should have a "molecular" component; that is a paper on new or modified polysaccharides should include structural information and characterization in addition to the usual studies of rheological properties and the like. A paper on a new, naturally occurring polysaccharide should include structural information, defining monosaccharide components and linkage sequence. Papers devoted wholly or partly to X-ray crystallographic studies, or to computational aspects (molecular mechanics or molecular orbital calculations, simulations via molecular dynamics), will be considered if they meet certain criteria. For computational papers the requirements are that the methods used be specified in sufficient detail to permit replication of the results, and that the conclusions be shown to have relevance to experimental observations - the authors'' own data or data from the literature. Specific directions for the presentation of X-ray data are given below under Results and "discussion".

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2,5-dihydroxybenzoic acid