Enhancing cytochrome P450-mediated non-natural cyclopropanation by mutation of a conserved second-shell residue.
Engineered 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.
['Gober|Joshua G|JG|', 'Rydeen|Amy E|AE|', 'Schwochert|Timothy D|TD|', "Gibson-O'Grady|Evan J|EJ|", 'Brustad|Eric M|EM|0000-0002-7031-5433']
D003521:Cyclopropanes D003577:Cytochrome P-450 Enzyme System D006418:Heme D050505:Mutant Proteins D020125:Mutation, Missense D010084:Oxidation-Reduction D015202:Protein Engineering D011994:Recombinant Proteins
cyclopropanation cytochrome P450 non-natural biocatalysis protein engineering second-shell mutation