Structural insights into the mechanism of the drastic changes in enzymatic activity of the cytochrome P450 vitamin D3 hydroxylase (CYP107BR1) caused by a mutation distant from the active site

Cytochromes P450 (P450s) are haem-containing enzymes that catalyze medically and industrially important oxidative reactions, and many P450s have been subjected to directed evolution and site-directed mutagenesis to improve their activity and substrate specificity. Nonetheless, in most cases the mechanism that leads to drastic changes in specific activity after the introduction of an amino-acid substitution distant from the active-site pocket is unclear. Here, two crystal structures of inactive mutants of the P450 vitamin D3 hydroxylase (Vdh), Vdh-F106V and Vdh-L348M, which were obtained in the course of protein-engineering experiments on Vdh, are reported. The overall structures of these mutants show an open conformation similar to that of wild-type Vdh (Vdh-WT), whereas a rearrangement of the common main-chain hydrogen bonds is observed in the CD-loop (residues 102 – 106), resulting in a more compactly folded CD-loop relative to that of Vdh-WT. The previously reported structures of Vdh-WT and of the highly active Vdh-T107A and Vdh-K1 mutants have a more stretched CD-loop, with partial formation of 310-helix-type hydrogen bonds, both in the open and closed states. Molecular-dynamics simulations also showed that the frequency of the 310-helix is significantly reduced in Vdh-F106V and Vdh-L348M. The closed conformation is crucial for substrate and ferredoxin binding to initiate the catalytic reaction of Vdh. Therefore, it is implied that the small local structural changes obs...
Source: Acta Crystallographica Section F - Category: Biochemistry Authors: Tags: crystal structure cytochrome P450 CYP vitamin D3 hydroxylase conformational changes research communications Source Type: research