期刊
NATURE CHEMISTRY
卷 10, 期 3, 页码 318-324出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/NCHEM.2927
关键词
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资金
- US Army Research Laboratory
- US Army Research Office [W911NF-14-1-0334, 66796-LS-RIP]
- National Science Foundation (NSF) under CAREER [CHE-1351991]
- David and Lucile Packard Foundation
- National Institutes of Health (NIH) National Cancer Institute [R00CA175399]
- Damon Runyon Cancer Research Foundation [DFS-08-14]
- NSF under the Center for Chemical Innovation Center for Selective C-H Functionalization [CHE-1700982]
- NIH Chemistry and Biology Interface Training Grant [T32 GM008720]
- Kwanjeong Educational Foundation
- NSF instrumentation grant [CHE-1048528]
- NATIONAL CANCER INSTITUTE [R00CA175399] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [DP2GM128199, T32GM008720] Funding Source: NIH RePORTER
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 cofactor 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 the 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-catalysed 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.
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