Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions. Reproducing these functions in a designed protein is the ultimate challenge to our understanding of them. Here, we present an artificial metallohydrolase, which has been shown by X-ray crystallography to contain two different metal ions—a Zn(II) ion, which is important for catalytic activity, and a Hg(II) ion, which provides structural stability. This metallohydrolase displays catalytic activity that compares well with several characteristic reactions of natural enzymes. It catalyses p-nitrophenyl acetate (pNPA) hydrolysis with an efficiency only ~100-fold less than that of human carbonic anhydrase (CA)II and at least 550-fold better than comparable synthetic complexes. Similarly, CO2 hydration occurs with an efficiency within ~500-fold of CAII. Although histidine residues in the absence of Zn(II) exhibit pNPA hydrolysis, miniscule apopeptide activity is observed for CO2 hydration. The kinetic and structural analysis of this first de novo designed hydrolytic metalloenzyme reveals necessary design features for future metalloenzymes containing one or more metals.
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V.L.P. acknowledges support from the National Institutes of Health (grant no. R01 ES0 12236), and M.L.Z. from the National Institutes of Health Chemistry–Biology Interface Training Program. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences (contract no. DE-AC02-06CH11357). Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant no. 085P1000817). J.A.S. is supported by the University of Michigan Center for Structural Biology.
The authors declare no competing financial interests.
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Zastrow, M., Peacock, A., Stuckey, J. et al. Hydrolytic catalysis and structural stabilization in a designed metalloprotein. Nature Chem 4, 118–123 (2012). https://doi.org/10.1038/nchem.1201
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