Cloning, Sequence Analysis, and Expression of Active Phrixothrix Railroad-Worms Luciferases:  Relationship between Bioluminescence Spectra and Primary Structures^,

Abstract

Phrixothrix railroad-worms emit yellow-green light through 11 pairs of lateral lanterns along the body and red light through two cephalic lanterns. The cDNAs for the lateral lanterns luciferase of Phrixothrix vivianii, which emit green light (lambda max= 542 nm), and for the head lanterns of P. hirtus, which emit the most red-shifted bioluminescence (lambda max= 628 nm) among luminescent beetles, were cloned. Positive clones which emitted green (PvGR: lambda max= 549 nm) and red (PhRE: lambda max= 622 nm) bioluminescence were isolated. The lucifereases coded by PvGR (545 amino acid residues) and PhRE (546 amino acid residues) cDNAs share 71% identity. PvGR and PhRE luciferases showed 50-55% and 46-49% identity with firefly luciferases, respectively, and 47-49% with click-beetle luciferases. PhRE luciferase has some unique residues which replace invariant residues in other beetle luciferases. The additional residue Arg 352 in PhRE, which is deleted in PvGR polypeptide, seems to be another important structural feature associated with red light production. As in the case of other railroad-worms and click-beetle luciferases studied, Phrixothrix luciferases do not undergo the typical red shift suffered by firefly luciferases upon decreasing pH, a property which might be related to the many amino acid residues shared in common between railroad-worm and click-beetle luciferase.

Active-Site Properties of Phrixotrix Railroad Worm Green and Red Bioluminescence-Eliciting Luciferases

“…Substrate Affinities and Catalytic Efficiency-The K M values for luciferin (Table 1) were lower than previously reported for the crude enzymes (29). Significantly, the value for PxRE luciferase is one of the lowest among the beetle luciferases (Table 1).…”

“…Because they are the only luciferases to produce red light, they have strong potential applications in a wide variety of bioanalytical contexts. Previously, we cloned the green-emitting luciferase from P. viviani and the red-emitting luciferase from P. hirtus railroad worms (29), which are 71% identical at the primary structure level. We used mutagenesis and chimerization studies based on a comparison of their primary structures to identify the structural determinants of the bioluminescence colours (30)(31)(32)(33).…”

Enhanced red‐emitting railroad worm luciferase for bioassays and bioimaging

“…PhRED cDNA was cloned, 15 and its sequence was optimized for mammalian expression in our laboratory. 10,11 The optimized cDNA in mammalian expression vector (pCMV-SLR), whose expression is driven by the CMV promoter, was used as the template for mutation.…”

“…14 Luciferase cDNAs have been cloned from many beetles, but only the one from the railroad worm Phrixothrix hirtus can produce naturally red-emitting luciferase (PhRED) and has an additional advantage of spectral pH insensitivity. 15 With the characterization of the firefly luciferase crystal structure (1LCI, 2D1Q, 2D1R, 2D1S, 2D1T), 16,17 site-directed mutagenesis becomes an effective way to explore the relationship between structure and function and to improve the characteristics (e.g., thermostability, activity, or spectrum) of beetle luciferases. A few red-emitting mutants have been constructed from natural yellow-green emitting firefly luciferases (e.g., Photinus pyralis (Ppy), [18][19][20][21][22][23][24][25] Luciola cruciata (Lcr), 26 Lampyris turkestanicus, 27 Luciola italica 12 ); however, their traits such as spectral pH-sensitivity and low activity or poor stability counteract their demanding applications.…”

Cis-regulatory hairpin-shaped mRNA encoding a reporter protein: catalytic sensing of nucleic acid sequence at single nucleotide resolution

“…The primer sequences used are summarized in Figure 5. [21][22][23] . They were PCR-amplified as above to incorporate an A-sensitive, T-sensitive or G-sensitive target-binding site, respectively, to give U-RNA SLG , A-RNA SLO and C-RNA SLR (Fig.…”