Enhancing cytochrome P450‐mediated non‐natural cyclopropanation by mutation of a conserved second‐shell residue

Joshua G. Gober; Amy E. Rydeen; Timothy D. Schwochert; Evan J. Gibson‐O'Grady; Eric M. Brustad

doi:10.1002/bit.26571

journal article Mar 06, 2018

Enhancing cytochrome P450‐mediated non‐natural cyclopropanation by mutation of a conserved second‐shell residue

Joshua G. Gober Amy E. Rydeen Timothy D. Schwochert Evan J. Gibson‐O'Grady Eric M. Brustad

Biotechnology and Bioengineering Vol. 115 No. 6 pp. 1416-1426 · Wiley

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Abstract

AbstractEngineered cytochrome P450s are emerging as powerful synthetic tools due to their ability catalyze non‐native metallocarbenoid and ‐nitrenoid insertion reactions. P450‐mediated cyclopropanation has garnered particular interest due to the high selectivity demonstrated by engineered scaffolds and their application towards the synthesis of therapeutic agents. We previously reported that mutation of a conserved, first‐shell heme‐ligating Cys to Ser led to significant improvements in cyclopropanation activity in a model enzyme, P450BM3h. Here, we demonstrate that mutation of a ubiquitously conserved second‐shell Phe (F393) to His or Ala, provides complementary increases in the P450 heme reduction potential and conversion to cyclopropanation products when compared to first‐shell Cys to Ser mutations. Furthermore, we show that these mutations confer improved non‐natural catalysis in 4 diverse P450 scaffolds.

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Details

Published: Mar 06, 2018
Vol/Issue: 115(6)
Pages: 1416-1426
License: View

Authors

J

Joshua G. Gober