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Glu311 and Arg337 Stabilize a Closed Active-site Conformation and Provide a Critical Catalytic Base and Countercation for Green Bioluminescence in Beetle Luciferases
journal contribution
posted on 2016-07-08, 00:00 authored by V. R. Viviani, A. Simões, V. R. Bevilaqua, G. V. M. Gabriel, F. G. C. Arnoldi, T. HiranoBeetle luciferases
elicit the emission of different bioluminescence
colors from green to red. Whereas firefly luciferases emit yellow-green
light and are pH-sensitive, undergoing a typical red-shift at acidic
pH and higher temperatures and in the presence of divalent heavy metals,
click beetle and railroadworm luciferases emit a wider range of colors
from green to red but are pH-independent. Despite many decades of
study, the structural determinants and mechanisms of bioluminescence
colors and pH sensitivity remain enigmatic. Here, through modeling
studies, site-directed mutagenesis, and spectral and kinetic studies
using recombinant luciferases from the three main families of bioluminescent
beetles that emit different colors of light (Macrolampis sp2 firefly, Phrixotrix hirtus railroadworm,
and Pyrearinus termitilluminans click beetle), we
investigated the role of E311 and R337 in bioluminescence color determination.
All mutations of these residues in firefly luciferase produced red
mutants, indicating that the preservation of opposite charges and
the lengths of the side chains of E311 and R337 are essential for
keeping a salt bridge that stabilizes a closed hydrophobic conformation favorable for green light emission.
Kinetic studies indicate that residue R337 is important for binding
luciferin and creating a positively charged environment around excited
oxyluciferin phenolate. In Pyrearinus green-emitting
luciferase, the R334A mutation causes a 27 nm red-shift, whereas in Phrixotrix red-emitting luciferase, the L334R mutation causes
a blue-shift that is no longer affected by guanidine. These results
provide compelling evidence that the presence of arginine at position
334 is essential for blue-shifting the emission spectra of most beetle
luciferases. Therefore, residues E311 and R337 play both structural
and catalytic roles in bioluminescence color determination, by stabilizing
a closed hydrophobic conformation favorable for green light emission,
and also providing a base to accept excited oxyluciferin phenol proton,
and a countercation to shield the negative charge of E311 and to stabilize
excited oxyluciferin phenolate, blue-shifting emission spectra in
most beetle luciferases.
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oxyluciferin phenol protonoxyluciferin phenolatebioluminescence colorsL 334R mutation causesE 311light emissionresidues E 311Macrolampis sp 2 fireflybeetle luciferasesBeetle Luciferases Beetle luciferasesArg 337 StabilizeR 337Phrixotrix hirtus railroadwormR 334A mutation causesCritical Catalytic Basebioluminescence color determinationresidue R 337
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