Designing artificial metalloenzymes with high activity through engineering secondary coordination sphere interactions

Conference Dates

September 15-19, 2019


Metalloenzymes can catalyze some the most difficult and important reactions in biology. Designing artificial metalloenzymes (ArMs) with similar structure and activity as native enzymes is an ultimate test of our knowledge about metalloenzymes and can result in new biocatalysts for practical applications [1]. Despite progress made, most ArMs display much lower activity than native enzymes. A critical step to advancing the field is fundamental understanding what it takes to not only confer, but also fine-tune the ArM activity to as high as native enzymes. Only after we can demonstrate the ability to modulate ArM activity at-will to rival (or surpass!) natural enzymes can the potential of ArMs be fully realized.

A key to unlocking the ArM potential is the observation that the same primary coordination sphere (PCS) of a protein metal center can display diverse functions and a range of activity levels, leading to the realization that interactions in the secondary coordination sphere (SCS) are critically important. However, the SCS interactions, compared to the PCS structures, are numerous, long-range, and weak, making them very difficult to reproduce in ArMs, due to the sheer complexity of protein three-dimensional structure.

In this presentation, I will provide recent examples from Lu group and collaborators to demonstrate that, while reproducing the PCS may be good enough to make structural models of metalloenzymes, careful design of the non-covalent SCS interactions is required to create functional metalloenzymes with high activity. In the first example, we have demonstrated that, while a design of CuB center next to the heme in myoglobin that structurally mimics the heme-copper center in oxidases resulted in minimal oxidase activity, engineering water and associated hydrogen bonding networks next the PCS resulted in a dramatic increase in the activity [2]. When combined with further engineering of the electron transfer interface, the ArM has a comparable activity as native oxidase in solution [3] and 10 times faster activity as an electrocatalyst for the oxygen reduction reaction [4]. In the second example, we have shown that, while a design of [4Fe-4S] cluster next to the heme center in cytochrome c peroxidase that structurally mimics the heme-[4Fe-4S] center in sulfite reductase resulted in no activity, engineering SCS interactions to tune the reduction potentials of the [4Fe-4S] cluster and to enhance the substrate binding affinity resulted in an ArM with 20% of the native enzyme activity [5]. In the third example, through careful design of hydrophobicity and hydrogen bonding networks around the PCS of Type 1 copper center in azurin, we have been able to tune its reduction potentials to span across the entire 2V physiological reduction potential range [6], something that has not been achieved by any single class of native metalloproteins. Finally, we have shown that, by introducing unnatural amino acids [7], non-native metal ions[8] and metallocofactors [9], we have been able to fine-tune the activity of ArMs even better than those of native enzymes.


1. a) Y. Lu, et al., Nature 460, 855 (2009); b) I. D. Petrik, J. Liu, Y. Lu, Curr. Opin. Chem. Biol. 19, 67 (2014);

c) E. N. Mirts, A. Bhagi-Damodaran, Y. Lu, Acc Chem. Res. (in press; DOI: 10.1021/acs.accounts.9b00011);

2. K. D. Miner, et al., Angew. Chem., Int. Ed. 51, 5589 (2012);

3. Y. Yu et al., J. Am. Chem. Soc. 137, 11570–11573 (2015);

4. S. Mukherjee et al., Nature Comm. 6, Article number: 8467; doi:10.1038/NCOMMS9467 (2015);

5. E, N. Mirts et al. Science 361, 1098-1101 (2018);

6. a) N. M. Marshall, et al., Nature 462, 113 (2009); b) P. Hosseinzadeh et al., Proc. Natl. Acad. Sci. USA 113, 262-267 (2016);

7. Y. Yu, et al., J. Am. Chem. Soc. 137, 4594-4597 (2015);

8. a) A. Bhagi-Damodaran et al., Nature Chem. 9, 257–263 (2017); J. H. Reed et al., J. Am. Chem. Soc. 139, 12209–12218 (2017);

9. a) A. Bhagi-Damodaran et al., J. Am. Chem. Soc. 136, 11882-11885 (2014); b) A. Bhagi-Damodaran et al., Proc. Natl. Acad. Sci. USA 115, 6195-6200 (2018).

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