Scholarlink

<p>Cytochrome P450s are haem-containing enzymes, catalysing the regio- and stereospecific oxidation of non-activated hydrocarbons. Among these, the bacterial P450BM3 is a promising biocatalyst due to its high enzymatic activity. Given the significant conformational flexibility of this enzyme, understanding protein-substrate interactions and associated structural dynamics are crucial for designing P450BM3-based biocatalysts. Herein, employing an X-ray free electron laser in combination with freeze-trap crystallography and spectroscopy techniques, we captured the intact structures of engineered P450BM3s in the initial stages of catalysis during styrene epoxidation, in the presence of a decoy molecule. We found that the iron reduction significantly altered the active-site orientation of styrene, driven by structural changes in surrounding helices and hydrogen-bonding networks. Oxygen binding to iron further stabilised its productive orientation, providing a molecular basis for the experimentally observed enzyme kinetics and enantioselectivities. This study reveals the substrate dynamics of a P450 enzyme, showcasing how changes in haem chemistry affect enzyme structure and substrate orientation.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

XFEL crystallography reveals catalytic cycle dynamics during non-native substrate oxidation by cytochrome P450BM3

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年3月12日

查看全文
  • Satoshi Nagao,
  • Wako Kuwano,
  • Takehiko Tosha,
  • Keitaro Yamashita,
  • Joshua Kyle Stanfield,
  • Chie Kasai,
  • Shinya Ariyasu,
  • Kunio Hirata,
  • Go Ueno,
  • Hironori Murakami,
  • Hideo Ago,
  • Masaki Yamamoto,
  • Osami Shoji,
  • Hiroshi Sugimoto,
  • Minoru Kubo

摘要

Cytochrome P450s are haem-containing enzymes, catalysing the regio- and stereospecific oxidation of non-activated hydrocarbons. Among these, the bacterial P450BM3 is a promising biocatalyst due to its high enzymatic activity. Given the significant conformational flexibility of this enzyme, understanding protein-substrate interactions and associated structural dynamics are crucial for designing P450BM3-based biocatalysts. Herein, employing an X-ray free electron laser in combination with freeze-trap crystallography and spectroscopy techniques, we captured the intact structures of engineered P450BM3s in the initial stages of catalysis during styrene epoxidation, in the presence of a decoy molecule. We found that the iron reduction significantly altered the active-site orientation of styrene, driven by structural changes in surrounding helices and hydrogen-bonding networks. Oxygen binding to iron further stabilised its productive orientation, providing a molecular basis for the experimentally observed enzyme kinetics and enantioselectivities. This study reveals the substrate dynamics of a P450 enzyme, showcasing how changes in haem chemistry affect enzyme structure and substrate orientation.