Abstract
We recently reported that a computationally designed catalyst nicknamed AlleyCat facilitates C–H proton abstraction in Kemp elimination at neutral pH in a selective and calcium-dependent fashion by a factor of approximately 100,000 (Korendovych et al. in Proc. Natl. Acad. Sci. USA 108:6823, 2011). Kemp elimination produced a colored product that can be easily read out, thus making AlleyCat a catalytically amplified metal sensor for calcium. Here we report that metal-binding EF-hand motifs in AlleyCat could be redesigned to incorporate trivalent metal ions without significant loss of catalytic activity. Mutation of a single neutral residue at position 9 of each of the EF-hands to glutamate results in almost a two orders of magnitude improvement of selectivity for trivalent metal ions over calcium. Development of this new lanthanide-dependent switchable Kemp eliminase, named CuSeCat EE, provides the foundation for further selectivity improvement and broadening the scope of the repertoire of metals for sensing. A concerted effort in the design of switchable enzymes has many environmental, sensing, and metal ion tracking applications.
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Domaille DW, Que EL, Chang CJ (2008) Nat Chem Biol 4:168–175
Luque de Castro MD, Herrera MC (2003) Biosens Bioelectron 18:279–294
Doi N, Yanagawa H (1999) FEBS Lett 453:305–307
Dwyer MA, Hellinga HW (2004) Curr Opin Chem Biol 14:495–504
Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY (1997) Nature 388:882–887
Zhu L, Anslyn E (2006) Angew Chem Int Ed 45:1190–1196
Ostermeier M (2009) Curr Opin Struct Biol 19:442–448
Yoon HJ, Kuwabara J, Kim J-H, Mirkin CA (2010) Science 330:66–69
Korendovych IV, Kulp DW, Wu Y, Cheng H, Roder H, DeGrado WF (2011) Proc Natl Acad Sci USA 108:6823–6827
Blommel PG, Fox BG (2007) Protein Expr Purif 55:53–68
Studier FW (2005) Protein Expr Purif 41:207–234
Rothlisberger D, Khersonsky O, Wollacott AM, Jiang L, DeChancie J, Betker J, Gallaher JL, Althoff EA, Zanghellini A, Dym O, Albeck S, Houk KN, Tawfik DS, Baker D (2008) Nature 453:190–195
Urbauer JL, Short JH, Dow LK, Wand AJ (1995) Biochemistry 34:8099–8109
Radivoyevitch T (2009) Biol Direct 4:49
Masino L, Martin SR, Bayley PM (2000) Protein Sci 9:1519–1529
Marley J, Lu M, Bracken C (2001) J Biomol NMR 20:71–75
Klee CB, Crouch TH, Richman PG (1980) Ann Rev Biochem 49:489–515
Bertini I, Gelis I, Katsaros N, Luchinat C, Provenzani A (2003) Biochemistry 42:8011–8021
Le Clainche L, Plancque G, Amekraz B, Moulin C, Pradines-Lecomte C, Peltier G, Vita C (2003) J Biol Inorg Chem 8:334–340
am Ende CW, Meng HY, Ye M, Pandey AK, Zondlo NJ (2010) ChemBioChem 11:1738–1747
Zondlo SC, Gao F, Zondlo NJ (2010) J Am Chem Soc 132:5619–5621
Moroz OV, Moroz YS, Mack KL, Wu Y, Olsen AB, Cheng H, McLaughlin JM, Raymond EA, Roder H, Korendovych IV (in preparation)
Drake SK, Falke JJ (1996) Biochemistry 35:1753–1760
Drake SK, Lee KL, Falke JJ (1996) Biochemistry 35:6697–6705
Falke JJ, Drake SK, Hazard AL, Peersen OB (1994) Q Rev Biophys 27:219–290
Ye YM, Lee H-W, Yang W, Shealy S, Yang JJ (2005) J Am Chem Soc 127:3743–3750
Chou JJ, Li S, Klee CB, Bax A (2001) Nat Struct Biol 8:990–997
O’Neil KT, DeGrado WF (1990) Trends Biochem Sci 15:59–64
Snyder EE, Buoscio BW, Falke JJ (1990) Biochemistry 29:3937–3943
Mulqueen P, Tingey JM, Horrocks WD Jr (1985) Biochemistry 24:6639–6645
Samish I, MacDermaid CM, Perez-Aguilar JS, Saven JG (2011) Annu Rev Phys Chem 62:129–149
DeGrado WF, Summa CM, Pavone V, Nastri F, Lombardi A (1999) Annu Rev Biochem 68:779–819
Das R, Baker D (2008) Annu Rev Biochem 77:363–382
Brustad EM, Arnold FH (2011) Curr Opin Chem Biol 15:201–210
Nitz M, Franz KJ, Maglathlin RL, Imperiali B (2003) ChemBioChem 4:272–276
Franz KJ, Nitz M, Imperiali B (2003) ChemBioChem 4:265–271
Nitz M, Sherawat M, Franz KJ, Peisach E, Allen KN, Imperiali B (2004) Angew Chem Int Ed 43:3682–3685
Acknowledgments
The authors thank Syracuse University for providing funds for this study, the Beckman Foundation for a scholarship for K.L.M., and Robert P. Doyle for use of his CD instrument.
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Mack, K.L., Moroz, O.V., Moroz, Y.S. et al. Reprogramming EF-hands for design of catalytically amplified lanthanide sensors. J Biol Inorg Chem 18, 411–418 (2013). https://doi.org/10.1007/s00775-013-0985-5
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DOI: https://doi.org/10.1007/s00775-013-0985-5