Crystals in Minutes

  • Author: Angewandte Chemie International Edition
  • Published Date: 22 March 2020
  • Source / Publisher: Angewandte Chemie International Edition/Wiley-VCH
  • Copyright: Wiley-VCH Verlag GmbH & Co. KGaA
thumbnail image: Crystals in Minutes

High-quality metalloenzyme crystals are important for the structure-based design of biocatalysts. However, preparing bulk quantities of metalloenzyme microcrystals is challenging. Cytochrome P450 102A1 (or P450BM3), for example, has a high catalytic activity and is a promising candidate for use as a versatile biocatalyst. When this enzyme is modified to change its reactivity, it can be almost impossible to obtain high‐quality crystals suitable for X‐ray crystallography.


Hiroshi Sugimoto, RIKEN SPring-8 Centre, Sayo, Japan, Science and Technology Agency, Tokyo, Japan, Osami Shoji, Nagoya University, Japan, and Science and Technology Agency, Japan, and colleagues have used a crystallization accelerator to obtain crystals of the haem domains of P450BM3 variants. The team used the diterpenoid derivative N‐abietoyl‐L‐tryptophan (AbiATrp)  to accelerate the crystallization of wild‐type, unmodified P450BM3. AbiATrp is a substrate analogue for the enzyme.


The resulting crystals were used as seeds to prepare microcrystals of a range of P450BM3 variants (example pictured above), including both substrate-free and substrate-analogue-bound variants, as well as heavily tagged variants and ones that contain synthetic metal complexes. Mixing the seeds of P50BM3-binding AbiATrp with the variants of P450BM3 resulted in microcrystallization within minutes (process pictured below). The microcrystals prepared by the method are suitable for analysis with X-ray free-electron lasers (XFEL), which allows the monitoring of reaction intermediates in real time.

 


 

 

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