Analytical Sciences

Cation-driven Optical Properties of Artificial Luciferases

Sung Bae KIM,*1 Simon MILLER,*2 Nobuhiro SUZUKI,*2 Toshiya SENDA,*2,*3 Ryo NISHIHARA,*4 and Koji SUZUKI*4

*1 National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba West, 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan
*2 Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan
*3 Department of Materials Structure Science, School of High Energy Accelerator Science, The Graduate University of Advanced Studies (Soken-dai), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan
*4 Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama, Kanagawa 223-8522, Japan

The present study demonstrates cation-driven optical properties of artificial luciferases (ALucs) from copepod luciferases, as an optical readout for bioanalysis. An assignment of the supersecondary structure code (SSC) of ALucs revealed that ALucs carry a helix-loop-helix structure, which appears at the same sites of the EF-hands of typical Ca²⁺-binding proteins. A mutagenesis study shows that the EF-hand-like structure is a pivotal site for enzymatic activity. The effects of 20 kinds of mono- and multivalent cations on ALuc activities were estimated with column-purified ALuc16. High pH values boost the ALuc activities with both the native coelenterazine and an analog called 6-pi-OH-CTZ. Multivalent cations, Ca(II), Mg(II), and Cr(VI), elevate and prolong the ALuc activities, whereas Co(II), Cu(II) and Pb(II) greatly hamper the ALuc activities. Ca(II) greatly prolongs the optical intensities, suggesting a contribution to the structural robustness of ALucs. The inhibitory effect of multivalent cations on the ALuc activities was utilized for creating dose-response curves. The intrinsic cation-driven selectivity and optical intensity of ALucs enable researchers to constitute de novo biosensors for multivalent cations.

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