Proteins can be evolved to accept unnatural substrates or to catalyze new chemical reactions. The process of evolution is guided by available structural information or by computational analysis. We developed a new simple method for evolution of enzymes that relies on differences in HSQC spectra of the protein in the presence and absence of substrate analog. This method has been successfully used to improve catalytic efficiency of calmodulin-based Kemp eliminase that operates through acid-base mechanism. Here we demonstrate that our approach can be applied to other protein scaffolds and reaction mechanisms. Recent work by the Reetz group showed that Kemp elimination can be promoted by coordination of Kemp substrate to the Fe(II)-heme center of cytochrome <i>P450-BM3 </i>and subsequent electron transfer between iron and Kemp. To test applicability of NMR guided evolution to redox-catalyzed Kemp elimination we chose to work with heme-containing protein myoglobin. Based on NMR analysis we introduce mutations in the vicinity of heme and screen for accelerated formation of a yellow 2-cyano-4-nitrophenol product. Our work presents further validation of novel method developed for design of artificial enzymes.

NMR guided approach to evolution of Myoglobin protein

256th National Meeting (2018)

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