Abstract
Renilla luciferase (RLUC) is a popular reporter enzyme for gene expression and biosensor applications, but it is an unstable enzyme whose catalytic mechanism remains to be elucidated. We titrated that one RLUC molecule can turn over about one hundred molecules of coelenterazine substrate. Mutagenesis of active site residue Pro220 extended the half-life of photon emission, yielding brighter luminescence in E. coli. Random mutagenesis uncovered two new mutations that stabilized and increased photon emission in vivo and in vitro, while ameliorating substrate inhibition. Further amended with a previously identified mutation, a new triple mutant showed a threefold improved kcat, as well as elevated luminescence in Arabidopsis. This advances the utility of RLUC as a reporter protein, biosensor, or resonance energy donor.
Highlights
Renilla luciferase (RLUC) is a cofactor-less, single subunit, blue light emitting luciferase isolated from the marine anthozoan Renilla reniformis (RLUC, E.C. number 1.13.12.5, luciferin-2-monooxygenase, decarboxylating) [1]
Aside from its utility as a reporter for gene expression assays, RLUC has found application in assays for protein interaction based on fragment complementation [2] and bioluminescence resonance energy transfer [3]
Previous docking simulations and mutagenesis of RLUC active site residues D120, E144, and H285, which are conserved with the catalytic triad of the dehalogenase LinB, suggested that they underlie the catalytic mechanism of RLUC
Summary
Address: 1Department of Biochemistry, Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996-0840, USA and 2The Rockefeller University, Molecular, Cell and Developmental Biology, 1230 York Avenue New York, NY 10065, USA. Published: 30 September 2008 Plant Methods 2008, 4:23 doi:10.1186/1746-4811-4-23
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