Conformational gating mechanism for processive catalysis of beta(1,3) -glucans

Processive catalysis is a fundamental molecular mechanism in nature to both build and dismantle complex biological polymers such as nucleic acids, proteins and carbohydrates, underpinning a broad spectrum of biotechnological applications. Here, we uncover a processive mechanism for the breakdown of β(1,3)-glucans, a carbohydrate class widespread across biological kingdoms from prokaryotes to eukaryotes. This mechanism involves a structurally dynamic active site, which adopts a tunnel-like conformation upon substrate binding, productively positioning the glycosidic bond for cleavage. Upon product release, the disruption of a pivotal salt bridge triggers the transition to an open conformation, establishing new polar interactions with the remnant substrate that are essential for translocation and the subsequent catalytic cycle. QM/MM calculations further reveal that this processive cleavage involves a non-canonical 4C1 reactive sugar puckering, a characteristic hitherto limited to certain exo-acting enzymes. Taken together, these findings establish the mechanistic basis for β(1,3)-glucan processive catalysis, demonstrating reaction steps from substrate recognition, tunnel formation, nucleophilic attack, intermediate state stabilization and conformational changes associated with product release and translocation. Ultimately, this work advances our understanding of microbial enzymatic systems for β(1,3)-glucan breakdown and metabolism, demonstrating that processive catalysis is a conserved evolutionary strategy across all major classes of β-glucans in nature.

持续催化（processive catalysis）是自然界中兼具合成与降解核酸、蛋白质、碳水化合物等复杂生物聚合物的核心分子机制，为广泛的生物技术应用奠定了支撑。本研究揭示了一种针对β(1,3)-葡聚糖的持续催化机制，这类碳水化合物广泛存在于从原核生物到真核生物的各个生物界中。该机制涉及一个结构动态变化的活性位点：在底物结合后，该位点会形成隧道状构象，将糖苷键精准定位以利于切割反应。当产物释放时，关键盐桥的断裂会触发活性位点向开放构象转变，并与残留底物建立新的极性相互作用，这一过程对于酶的移位及后续催化循环至关重要。量子力学/分子力学（QM/MM）计算进一步揭示，该持续切割过程涉及一种非典型的4C1构象反应性糖环褶皱，这一特征此前仅见于部分外切酶。综上，本研究明确了β(1,3)-葡聚糖持续催化的机制基础，完整阐释了从底物识别、隧道形成、亲核攻击、中间态稳定到产物释放与移位相关构象变化的全部反应步骤。最终，本研究加深了我们对微生物介导β(1,3)-葡聚糖降解与代谢的酶促系统的认知，并证实持续催化是自然界中所有主要β-葡聚糖类物质所共有的进化策略。