How the unique luciferase of
Phrixothrix hirtus
(PxRE) railroad worm catalyzes the emission of red bioluminescence using the same luciferin of fireflies, remains a mystery. Although PxRE luciferase is a very attractive tool for bioanalysis and bioimaging in hemoglobin rich tissues, it displays lower quantum yield (15%) when compared to green emitting luciferases (>40%). To identify which parts of PxRE luciferin binding site (LBS) determine bioluminescence color, and to develop brighter and more red-shifted emitting luciferases, we compared the effects of site-directed mutagenesis and of larger 6′-substituted aminoluciferin analogues (6′-morpholino- and 6′-pyrrolidinyl-LH) on the bioluminescence properties of PxRE and green-yellow emitting beetle luciferases. The effects of mutations in the benzothiazolyl and thiazolyl parts of PxRE LBS on the K
M
and catalytic efficiencies, indicated their importance for luciferin binding and catalysis. However, the absence of effects on the bioluminescence spectrum indicated a less interactive LBS in PxRE during light emission. Mutations at the bottom of LBS of PxRE blue-shifted the spectra and increased catalytic efficiency, suggesting that lack of interactions of this part of LBS with excited oxyluciferin phenolate underlie red light emission. The much higher bioluminescence activity and red-shifted spectra of PxRE luciferase with 6′-morpholino- (634 nm) and 6′-pyrrolidinyl-luciferins (644 nm), when compared to other beetle luciferases, revealed a larger luciferin phenolate binding pocket. The size and orientation of the side-chains of L/I/H348 are critical for amino-analogues accommodation and modulate bioluminescence color, affecting the interactions and mobility of excited oxyluciferin phenolate. The
PxRE
luciferase and 6′-aminoluciferins provide potential far-red combinations for bioimaging applications.
The control region (CR) or A + T-rich region in Coleoptera mt genome is poorly characterized, including the Elateroidea bioluminescent species. Here, we provided the first attempt to characterize and compare the structure and organization of the CR of different species within Elateridae. We also revisited some sequenced Coleoptera CR and observed consensus T-stretches, non-conserved sequences near the stem-loop and unusual inner tRNAs-like sequences. All these features are probably involved in the replication start of the mt genome. The phylogenetic relationships in Elateridae bioluminescent groups using partial sequence of CR showed the monophyly of Pyrearinus pumilus group and Pyrearinus as a polyphyletic genus, corroborating our previous results. The wider genetic variation obtained by CR analysis could separate two different lineages that occur within P. termitilluminans populations. In Elateridae, the CR exhibited high polymorphism within and between populations, which was also observed in other Coleoptera species, suggesting that the CR could be described as a suitable molecular marker to be applied in phylogenetic and phylogeographic studies.
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