Presentation Title

Development and Optimization of Artificial Metalloenzymes

Faculty Mentor

John F. Hartwig

Start Date

17-11-2018 12:30 PM

End Date

17-11-2018 2:30 PM

Location

CREVELING 27

Session

POSTER 2

Type of Presentation

Poster

Subject Area

physical_mathematical_sciences

Abstract

Organometallic complexes consisting of transition metal elements and distinct ligands have been utilized to catalyze important transformations. With catalytically active organometallic sites integrated into a protein matrix, artificial metalloenzymes could potentially extend the reaction scope and the performance of organometallic catalysts. Moreover, fine-tuning substrate recognition and stereoselectivity from the second ligand sphere provided by the protein offers new possibilities of catalyst control. We have primarily focused on the modification of Sulfolobus solfataricus P450 enzyme (CYP119), which naturally contains heme in its active site. After expressing CYP119 mutants (M69C, A209C, G210C, G317C) with cysteine near the active site, we tested out the bioconjugation of terpyridine acetamide-Mn and terpyridine maleimide-Mn cofactors. MS and UV-Vis evidence showed that while acetamide cofactors showed no bioconjugation, maleimide ones were successfully anchored to the scaffold. To further develop the optimum metalloprotein complex, we expanded the mutant library by combinatorial codon mutagenesis strategy and efficiently expressed mutants with 19 out of 20 amino acids at M69X. Meanwhile, we also tested dative linkage of porphyrin iridium complexes to our protein scaffold, since iridium complexes are shown to be excellent catalysts in C-H activation from our lab's past researches. The removal of hemin from CYP119 would enable the reconstitution of apo-protein with artificial metalloporphyrins. To enhance the iridium metallation, we synthesized electron-deficient ligand such as fluorinated and NO2 substituted porphyrins. Aside from CYP119, we have also expressed mono-cysteine mutants of BamB, a beta-barrel protein with large enough cavity for artificial cofactors. Successful anchoring of terpyridine maleimide ligand onto E61C mutant has been observed. Through different approaches, we have obtained a variety of protein scaffolds, unnatural cofactors, successful bioconjugation examples and substrates for testing out catalytic activities. The subsequent screening would shed light on the potential of artificial metalloenzymes and the influence of variables on their selectivity and performance.

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Nov 17th, 12:30 PM Nov 17th, 2:30 PM

Development and Optimization of Artificial Metalloenzymes

CREVELING 27

Organometallic complexes consisting of transition metal elements and distinct ligands have been utilized to catalyze important transformations. With catalytically active organometallic sites integrated into a protein matrix, artificial metalloenzymes could potentially extend the reaction scope and the performance of organometallic catalysts. Moreover, fine-tuning substrate recognition and stereoselectivity from the second ligand sphere provided by the protein offers new possibilities of catalyst control. We have primarily focused on the modification of Sulfolobus solfataricus P450 enzyme (CYP119), which naturally contains heme in its active site. After expressing CYP119 mutants (M69C, A209C, G210C, G317C) with cysteine near the active site, we tested out the bioconjugation of terpyridine acetamide-Mn and terpyridine maleimide-Mn cofactors. MS and UV-Vis evidence showed that while acetamide cofactors showed no bioconjugation, maleimide ones were successfully anchored to the scaffold. To further develop the optimum metalloprotein complex, we expanded the mutant library by combinatorial codon mutagenesis strategy and efficiently expressed mutants with 19 out of 20 amino acids at M69X. Meanwhile, we also tested dative linkage of porphyrin iridium complexes to our protein scaffold, since iridium complexes are shown to be excellent catalysts in C-H activation from our lab's past researches. The removal of hemin from CYP119 would enable the reconstitution of apo-protein with artificial metalloporphyrins. To enhance the iridium metallation, we synthesized electron-deficient ligand such as fluorinated and NO2 substituted porphyrins. Aside from CYP119, we have also expressed mono-cysteine mutants of BamB, a beta-barrel protein with large enough cavity for artificial cofactors. Successful anchoring of terpyridine maleimide ligand onto E61C mutant has been observed. Through different approaches, we have obtained a variety of protein scaffolds, unnatural cofactors, successful bioconjugation examples and substrates for testing out catalytic activities. The subsequent screening would shed light on the potential of artificial metalloenzymes and the influence of variables on their selectivity and performance.