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<p>Enzymes undergo dynamic conformational changes during catalysis, yet conventional high-resolution structural methods typically capture only the most stable states. Here, we address this gap using rapid UV photolysis of a chemically caged substrate with cryogenic time-resolved electron microscopy (cryo-TREM). We elucidate the catalytic mechanism of GtrB, a membrane-bound glycosyltransferase that transfers glucose from UDP-glucose to the lipid carrier undecaprenyl phosphate. We visualized how GtrB, which has an active site ~15 Å from the membrane, transitions during the catalytic cycle to move each substrate in proximity for catalysis. From a single dataset, we resolved distinct conformational states: the initial substrate-bound state, a catalytically poised intermediate, and the product-bound state. Through molecular dynamics simulations and biochemical analyses, we identify coordinated movements within the active site that drive catalysis. These findings provide a molecular framework for understanding how glycosyltransferases function and highlight a broadly applicable strategy for capturing dynamic enzymatic states in native-like environments.</p>

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Mechanistic snapshots of lipid-linked sugar transfer

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月6日

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  • Ryan T. Morgan,
  • Stefano Motta,
  • Eva Gil-Iturbe,
  • Biddut Bhattacharjee,
  • Mohammad T. Anwar,
  • Giovanni Di Muccio,
  • Alice Romagnoli,
  • Bedangshu Mishra,
  • Khuram U. Ashraf,
  • Injin Bang,
  • Daniele Di Marino,
  • Todd L. Lowary,
  • Matthias Quick,
  • Vasileios I. Petrou,
  • Michael H. B. Stowell,
  • Rie Nygaard,
  • Filippo Mancia

摘要

Enzymes undergo dynamic conformational changes during catalysis, yet conventional high-resolution structural methods typically capture only the most stable states. Here, we address this gap using rapid UV photolysis of a chemically caged substrate with cryogenic time-resolved electron microscopy (cryo-TREM). We elucidate the catalytic mechanism of GtrB, a membrane-bound glycosyltransferase that transfers glucose from UDP-glucose to the lipid carrier undecaprenyl phosphate. We visualized how GtrB, which has an active site ~15 Å from the membrane, transitions during the catalytic cycle to move each substrate in proximity for catalysis. From a single dataset, we resolved distinct conformational states: the initial substrate-bound state, a catalytically poised intermediate, and the product-bound state. Through molecular dynamics simulations and biochemical analyses, we identify coordinated movements within the active site that drive catalysis. These findings provide a molecular framework for understanding how glycosyltransferases function and highlight a broadly applicable strategy for capturing dynamic enzymatic states in native-like environments.