title: Reprogramming EF-hands for design of catalytically amplified lanthanide sensors.
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.
authors: Department of Chemistry, Syracuse University, Syracuse, NY 13244, USA.
authors: Korrie L
authors: KL
authors: Mack
authors: Olesia V
authors: OV
authors: Moroz
authors: Yurii S
authors: YS
authors: Moroz
authors: Alissa B
authors: AB
authors: Olsen
authors: Jaclyn M
authors: JM
authors: McLaughlin
authors: Ivan V
authors: IV
authors: Korendovych
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D003198:Computer Simulation; D015195:Drug Design; D020832:EF Hand Motifs / Q000235:genetics*; D007700:Kinetics; D028581:Lanthanoid Series Elements / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015335:Molecular Probes / Q000235:genetics; D011485:Protein Binding
chemical_list: D028581:Lanthanoid Series Elements; D015335:Molecular Probes