Evolving artificial metalloenzymes via random mutagenesis

Abstract

Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free co-factor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. This improved selectivity carried over to other dirhodium catalyzed transformations, including N- H, S-H and Si-H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.

Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid

“…[12][13][14] In this report, we demonstrate that directed evolution is also am eans to boost the proficiency of an ovel class of designer enzymes,those that feature an unnatural amino acid as ac atalytic residue. [15][16][17] Such designer catalysts mimic natural enzymes that employ posttranslational modifications of active site residues to install uniquely reactive functionalities to promote their target reactions (i.e.formylglycine in type-I-sulfatases). [18][19][20] Designer enzymes that make use of catalytic unnatural amino acids are also distinct from protein engineering efforts,i nw hich genetic-code expansion strategies [21,22] have been used to install non-standard side chains to improve/alter hydrophobic packing or substrate recognition.…”

Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid

“…In this report, we demonstrate that directed evolution is also a means to boost the proficiency of a novel class of designer enzymes, those that feature an unnatural amino acid as a catalytic residue . Such designer catalysts mimic natural enzymes that employ posttranslational modifications of active site residues to install uniquely reactive functionalities to promote their target reactions (i.e.…”

Selective C H bond functionalization with engineered heme proteins: new tools to generate complexity

“…The creation of artificial metalloenzymes which contain noble metal complexes [45,46], including the replacement of the heme group in heme proteins with porphyrins containing alternative metals, is one strategy to expand the scope of reactions accessible to enzymes. Replacing the iron-porphyrin cofactor with an iridium-porphyrin creates artificial metalloenzymes which install a new C-C bond in place of an sp 3 C-H bond (Figure 4a).…”