Pyrearinus termitilluminans larval click beetle luciferase: active site properties, structure and function relationships and comparison with other beetle luciferases

Abstract

Several beetle luciferases have been cloned and sequenced. However, most studies on structure and function relationships and bioanalytical applications were done with firefly luciferases, which are pH sensitive. Several years ago we cloned Pyrearinus termitilluminans larval click beetle luciferase, which displays the most blue-shifted bioluminescence among beetle luciferases and is pH insensitive. This enzyme was expressed in E. coli, purified, and its properties investigated. This luciferase shows slower luminescence kinetics, K(M) values comparable to other beetle luciferases and high catalytic constant. Fluorescence studies with 8-anilino-1-naphtalene-sulfonic acid (1,8-ANS) and modeling studies suggest that the luciferin binding site of this luciferase is very hydrophobic, supporting the solvent and orientation polarizability effects as determining mechanisms for bioluminescence colors. Although pH insensitive in the range between pH 6-8, at pH 10 this luciferase displays a remarkable red-shift and broadening of the bioluminescence spectrum. Modeling studies suggest that the residue C312 may play an important role in bioluminescence color modulation. Compared to other beetle luciferases, Pyrearinus termitilluminans luciferase also displays higher thermostability and sustained luminescence in a bacterial cell environment, which makes this luciferase particularly suitable for in vivo cell analysis and bioimaging.

Enhanced Beetle Luciferase for High-Resolution Bioluminescence Imaging

“…Therefore, these kinetic properties may not contributed to the high light output of ELuc in living cells. In contrast, the decay constant of the luminescence of the wild-type luciferase is higher than that of FLuc [6]. When we measured the luminescence kinetics of purified FLuc protein by adding ATP and Mg 2+ as cofactors and D-luciferin, the luminescence displayed flash-like kinetics (Figure 1E), as descried previously [10].…”

“…The sustaining luminescence kinetics of FLuc are partly attributed to coenzyme A (CoA), a vestigial substrate of beetle luciferase included with the PicaGene reagent, by which CoA removes from the FLuc active site the inhibitors that are produced by luciferin–luciferase reaction [15], [16]. Similarly, CoA also seems to participate in the long-lasting light production of ELuc, as discussed by Silva Neto et al [6]. In contrast to the light output from living cells, the resulting bioluminescence intensity of purified ELuc protein (normalized to the amount of protein) was unexpectedly slightly lower than that of FLuc, (Figure S3B), suggesting that the brighter luminescent signal from viable cells is not simply attributable to the enzymatic activity of the lucifearase.…”

“…Recently the purified enzyme has been shown to display high catalytic properties and thermostability in vitro among the pH-insensitive luciferases, producing to bright signals [6]. To improve the expression of P. termitilluminans luciferase in mammalian cells, we optimized codons of its cDNA sequence for mammalian expression and deleted putative transcription factor binding sites within the cDNA, without changing the deduced amino acid sequence (Figure S1).…”

“…Recently, several physicochemical properties of recombinant wild-type P. termitilluminans luciferase (the amino acid sequence of which is the same as that of ELuc, with the exception of a deletion of the duplicated first methionine, as described in the Materials and Methods section) have been determined, and these have been compared with those of other beetle luciferases, including FLuc [6]. The Michaelis–Menten constant ( K m ) of the wild-type P. termitilluminans luciferase for D-luciferin is rather higher than that of FLuc, whereas their K m values for adenosine triphosphate (ATP) are almost identical.…”

Comparison of the thermostability of recombinant luciferases from Brazilian bioluminescent beetles: Relationship with kinetics and bioluminescence colours

“…Comparing the K M values for luciferin and ATP of the wild‐type luciferases and the half‐life (Table ) we found that P . termitilluminans , which shows the highest thermostability, displayed high values of K M for ATP and luciferin . Similarly, P. vivianii green‐emitting luciferase, which is the most stable among railroad worm luciferases, also showed high K M values for both substrates, especially for luciferin which was one of the highest reported for beetle luciferases.…”