The variable structural flexibility of the Bacillus circulans β-galactosidase isoforms determines their unique functionalities

IF 4.4 2区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Structure Pub Date : 2024-09-30 DOI:10.1016/j.str.2024.09.005

Michaela Hovorková, Barbora Kaščáková, Lucie Petrásková, Petra Havlíčková, Jiří Nováček, Daniel Pinkas, Zdenko Gardian, Vladimír Křen, Pavla Bojarová, Ivana Kutá Smatanová

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Abstract

β-Galactosidase from Bacillus circulans ATCC 31382 (BgaD) is a biotechnologically important enzyme for the synthesis of β-galactooligosaccharides (GOS). Among its four isoforms, isoform A (BgaD-A) has distinct synthetic properties. Here, we present cryoelectron microscopy (cryo-EM) structures of BgaD-A and compare them with the known X-ray crystal structure of isoform D (BgaD-D), revealing substantial structural divergences between the two isoforms. In contrast to BgaD-D, BgaD-A features a flexible Big-4 domain and another enigmatic domain. The newly identified flexible region in BgaD-A is termed as “barrier domain 8,” and serves as a barricade, obstructing the access of longer oligosaccharide substrates into the active site of BgaD-A. The transgalactosylation reactions catalyzed by both isoforms revealed that BgaD-A has a higher selectivity than BgaD-D in the earlier stages of the reaction and is prevailingly directed to shorter galactooligosaccharides. This study improves our understanding of the structural determinants governing β-galactosidase catalysis, with implications for tailored GOS production.

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环状芽孢杆菌 β-半乳糖苷酶异构体的结构灵活性决定了它们的独特功能

环状芽孢杆菌（Bacillus circulans）ATCC 31382 的 β-半乳糖苷酶（BgaD）是合成 β-半乳寡糖（GOS）的一种重要生物技术酶。在其四种同工酶中，同工酶 A（BgaD-A）具有独特的合成特性。在这里，我们展示了 BgaD-A 的冷冻电子显微镜（cryo-EM）结构，并将其与已知的同工型 D（BgaD-D）的 X 射线晶体结构进行了比较，发现这两种同工型之间存在着很大的结构差异。与 BgaD-D 不同，BgaD-A 具有一个灵活的 Big-4 结构域和另一个神秘的结构域。在 BgaD-A 中新发现的柔性区域被称为 "障碍结构域 8"，它起着屏障的作用，阻碍较长的寡糖底物进入 BgaD-A 的活性位点。两种异构体催化的转半乳糖基化反应表明，BgaD-A在反应早期阶段的选择性比BgaD-D高，并且主要针对较短的半乳糖寡糖。这项研究加深了我们对支配β-半乳糖苷酶催化作用的结构决定因素的了解，对定制化 GOS 生产具有重要意义。

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来源期刊

Structure 生物-生化与分子生物学

CiteScore

8.90

自引率

1.80%

发文量

155

审稿时长

3-8 weeks

期刊介绍： Structure aims to publish papers of exceptional interest in the field of structural biology. The journal strives to be essential reading for structural biologists, as well as biologists and biochemists that are interested in macromolecular structure and function. Structure strongly encourages the submission of manuscripts that present structural and molecular insights into biological function and mechanism. Other reports that address fundamental questions in structural biology, such as structure-based examinations of protein evolution, folding, and/or design, will also be considered. We will consider the application of any method, experimental or computational, at high or low resolution, to conduct structural investigations, as long as the method is appropriate for the biological, functional, and mechanistic question(s) being addressed. Likewise, reports describing single-molecule analysis of biological mechanisms are welcome. In general, the editors encourage submission of experimental structural studies that are enriched by an analysis of structure-activity relationships and will not consider studies that solely report structural information unless the structure or analysis is of exceptional and broad interest. Studies reporting only homology models, de novo models, or molecular dynamics simulations are also discouraged unless the models are informed by or validated by novel experimental data; rationalization of a large body of existing experimental evidence and making testable predictions based on a model or simulation is often not considered sufficient.