Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

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

Structural insights into the mechanism of the drastic changes in enzymatic activity of the... 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 observed in this study might disrupt the conformational transition from the open to the closed state, thereby leading to a complete loss of vitamin D3 hydroxylase activity. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Crystallographica Section F Wiley

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

Download PDF
10 pages

Loading next page...
 
/lp/wiley/structural-insights-into-the-mechanism-of-the-drastic-changes-in-3xYD2WU3rm

References (54)

Publisher
Wiley
Copyright
Copyright © 2017 Wiley Subscription Services
ISSN
2053-230X
eISSN
2053-230X
DOI
10.1107/S2053230X17004782
pmid
28471358
Publisher site
See Article on Publisher Site

Abstract

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 observed in this study might disrupt the conformational transition from the open to the closed state, thereby leading to a complete loss of vitamin D3 hydroxylase activity.

Journal

Acta Crystallographica Section F – Wiley

Published: Jan 1, 2017

Keywords: ; ; ; ;

There are no references for this article.