4.5 Article

The structure of CYP101D2 unveils a potential path for substrate entry into the active site

Journal

BIOCHEMICAL JOURNAL
Volume 433, Issue -, Pages 85-93

Publisher

PORTLAND PRESS LTD
DOI: 10.1042/BJ20101017

Keywords

access channel; crystal structure; cytochrome P450; multi-step binding mechanism; Novosphingobium aromaticivorans DSM12444; open conformation

Funding

  1. Ministry of Science and Technology of China [2007CB914301]
  2. Tianjin Municipal Science and Technology Commission [08SYSYTC00200]
  3. Higher Education Funding Council for England

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The cytochrome P450 CYP101D2 from Novosphingobium aromaticivorans DSM12444 is closely related to CYP101D1 from the same bacterium and to P450cam (CYP101A1) from Pseudomonas putida. All three are capable of oxidizing camphor stereoselectively to 5-exo-hydroxycamphor. The crystal structure of CYP101D2 revealed that the likely ferredoxin-binding site on the proximal face is largely positively charged, similar to that of CYP101D1. However, both the native and camphor-soaked forms of CYP101D2 had open conformations with an access channel. In the active site of the camphor-soaked form, the camphor carbonyl interacted with the haem-iron-bound water. Two other potential camphor-binding sites were also identified from electron densities in the camphor-soaked structure: one located in the access channel, flanked by the B/C and F/G loops and the I helix, and the other in a cavity on the surface of the enzyme near the F helix side of the F/G loop. The observed open structures may be conformers of the CYP101D2 enzyme that enable the substrate to enter the buried active site via a conformational selection mechanism. The second and third binding sites may be intermediate locations of substrate entry and translocation into the active site, and provide insight into a multi-step substrate-binding mechanism.

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