Engineering the metal sensitive sites in Macrolampis sp2 firefly luciferase and use as a novel bioluminescent ratiometric biosensor for heavy metals

Abstract

Most luminescent biosensors for heavy metals are fluorescent and rely on intensity measurements, whereas a few are ratiometric and rely on spectral changes. Bioluminescent biosensors for heavy metals are less common. Firefly luciferases have been coupled to responsive promoters for mercury and arsenium, and used as light on biosensors. Firefly luciferase bioluminescence spectrum is naturally sensitive to heavy metal cations such as zinc and mercury and to pH. Although pH sensitivity of firefly luciferases was shown to be useful for ratiometric estimation of intracellular pH, its potential use for ratiometric estimation of heavy metals was never considered. Using the yellow-emitting Macrolampis sp2 firefly luciferase and site-directed mutagenesis, we show that the residues H310 and E354 constitute two critical sites for metal sensitivity that can be engineered to increase sensitivity to zinc, nickel, and mercury. A linear relationship between cation concentration and the ratio of bioluminescence intensities at 550 and 610 nm allowed, for the first time, the ratiometric estimation of heavy metals concentrations down to 0.10 mM, demonstrating the potential applicability of firefly luciferases as enzymatic and intracellular ratiometric metal biosensors.

The proton and metal binding sites responsible for the pH-dependent green-red bioluminescence color tuning in firefly luciferases

“…Since in Cratomorphus distinctus and Photinus pyralis luciferases E354 is at hydrogen bonding distance to H310, potentially making a salt bridge, and histidines have strong affinity to divalent metals, we also investigated whether the mutation of H310 affects metal sensitivity. The mutant H310A 8 , in which the metal chelating imidazole side-chain is removed, indeed was much less sensitive to metal ions, indicating that H310 is also important to bind metal ions such as Zn 2+ . These results prompted us to investigate the effect of other substitutions of H310 and N354 by residues with metal chelating side-chains such as His, Cys or Glu on the metal-sensitivity of Macrolampis firefly luciferase, resulting in a recently published paper in which we produced mutants with different sensitivities to Zn 2+ , Hg 2+ Cd 2+ and Ni 2+ which could be used to ratiometrically estimate these metal concentrations 8 .…”

“…4 ). The imidazole and carboxylate side chains of residues at positions 310 and 354 are responsible for modulating the sensitivity of Macrolampis and Cratomorphus firefly luciferases to metal ions such as nickel and zinc 8 . The interaction of the divalent metal ions with these glutamates in most firefly luciferases disrupt the salt bridges between E311/R337 and H310/E354, unshielding the positive charges of H310 and especially of R337, polarizing the environment (Fig.…”

“…All beetle luciferases have E311, and in most of them the negative charge of E311 carboxylate is counterbalanced by the positive charge of guanidinium group of residue R337 (only Phrixotrix luciferases do not). According to CTIL (charger transfer induced luminescence) mechanism, during the chemiexcitation step, the phenolic hydroxyl group of the dioxetanone intermediate must be necessarily deprotonated to give phenolate, in order to induce intramolecular charge transfer, promoting its decomposition into carbon dioxide and the excited singlet oxyluciferin which decays with emission of light 8 . The carboxylate of E311, assisted by a water molecule, may play the essential role of catalytic base for proton abstraction of the phenolic hydroxyl group of the dioxetanone intermediate, generating excited oxyluciferin phenolate during the chemiexcitation step.…”

“…Although many studies during the last decades have attempted to elucidate the mechanisms of bioluminescence color determination in beetle luciferases, the specific structural targets and mechanism of pH-sensitivity have remained elusive. Because firefly luciferases and their genes are widely used as bioanalytical reagents, reporter genes and biosensors 5 , 6 , and more recently are emerging as novel intracellular ratiometric biosensors for pH and heavy metals ions 7 , 8 , there is a lot of interest to better understand the origin of pH-sensitivity.…”

“…Considering that H310, E311 and E354 display both prototropic and metal chelating side-chains, we decided to investigate whether they are involved in metal and pH-sensitivity. The mutation of H310 and N354 by other residues with metal chelating groups in Macrolampis firefly luciferase was recently shown to affect the metal-sensitivity in firefly luciferases 8 , indicating that this region is indeed critical for metal-sensitivity in firefly luciferases. Here we compared the effect of natural and artificial substitutions of these residues on metal- and pH-sensitivities of three Brazilian firefly luciferases displaying different bioluminescence colors ( Amydetes vivianii : 539 nm; Cratomorphus distinctus : 548 nm and Macrolampis sp2: 569 nm; Fig.…”