Luciferase, bacterial bioluminescence

Fig. 2.1 Mechanism of the bacterial bioluminescence reaction. The molecule of FMNH2 is deprotonated at N1 when bound to a luciferase molecule, which is then readily peroxidized at C4a to form Intermediate A. Intermediate A reacts with a fatty aldehyde (such as dodecanal and tetradecanal) to form Intermediate B. Intermediate B decomposes and yields the excited state of 4a-hydroxyflavin (Intermediate C) and a fatty acid. Light (Amax 490 nm) is emitted when the excited state of C falls to the ground state. The ground state C decomposes into FMN plus H2O. All the intermediates (A, B, and C) are luciferase-bound forms. The FMN formed can be reduced to FMNH2 in the presence of FMN reductase and NADH.

The reported quantum yields of the long-chain aldehydes in the luminescence reaction catalyzed by P. fischeri luciferase are 0.1 for dodecanal with the standard I (Lee, 1972) 0.13 for decanal with the standard I (McCapra and Hysert, 1973) and 0.15-0.16 for decanal, dodecanal and tetradecanal with the standard III (Shimomura et al., 1972). Thus, the quantum yield of long-chain aldehydes in the bacterial bioluminescence reaction appears to be in the range of 0.10-0.16. 

Eckstein, J. W., Hastings, J. W., and Ghisla, S. (1993). Mechanism of bacterial bioluminescence. 4a,5-Dihydroflavin analogs as models for luciferase hydroperoxide intermediates and the effect of substituents at the 8-position of flavin on luciferase kinetics. Biochemistry 32 404 111. 

Eley, M., et al. (1970). Bacterial bioluminescence. Comparisons of bioluminescence emission spectra, the fluorescence of luciferase reaction mixtures, and the fluorescence of flavin cations. Biochemistry 9 2902-2908. 

Kurfuerst, M., Hastings, J. W., Ghisla, S., and Macheroux, P. (1984). Identification of the luciferase-bound flavin-4a-hydroxide as the primary emitter in the bacterial bioluminescence reaction. In Bray, R. C., et al. (eds.), Flavins Flavoproteins, Proc. Int. Symp. 8th, pp. 657-667. de Gruyter, Berlin. 

Riendeau, D., and Meighen, E. A. (1980). Co-induction of fatty acid reductase and luciferase during development of bacterial bioluminescence. J. Biol. Chem. 255 12060-12065. 

Waddle, J., and Baldwin, T. O. (1991). Individual a and p subunits of bacterial luciferase exhibit bioluminescence activity. Biochem. Biophys. Res. Commun. 178 1188-1193. 

Bacterial bioluminescence, 30-46 factors required, 31 general scheme, 32 in vivo luminescence, 41 luminescence reaction, 37, 38 Bacterial luciferase, 33-35, 343 assay, 39 cloning, 34 crystal structure, 34 extraction and purification, 34 inactivation, 34, 35 molecular weight, 34 properties, 34 storage, 35 subunits, 34... 

The bacterial bioluminescent reaction is also catalyzed by a luciferase (EC 1.14.14.3) isolated from marine bacteria. The four most studied types are Vibrio harveyi, Vibrio fischeri, Photobacterium phosphoreum and Photobacterium leiognathi18, 19. In these different luminescent bacteria the... 

An important phenomenon, now called quorum sensing, was discovered from studies of bacterial bioluminescence, in which it was found that growth and luminescence are controlled separately.22 After inoculating a culture into fresh medium, growth is exponential with no lag, but the amount of luciferase remains constant for the first three hours, after which its synthesis and light emission increase very, very rapidly (Fig. 6). This was shown to be due to the production and release into the medium of a substance that we named autoinducer upon reaching a critical concentration, it induces the synthesis of luciferase and other proteins involved in the bioluminescence. Eberhard and colleagues determined the structure to be a homoserine lactone and synthesized it.23... 

Paralogues of eubacterial riboflavin synthase (designated as lumazine protein, yellow fluorescent protein, and blue fluorescent protein) without enzymatic activity have been isolated from several luminescent bacteria. These proteins are highly fluorescent and are believed to modulate to serve as optical transponders that modulate the spectral characteristics of bacterial bioluminescence.Energy is believed to be transferred from activated bacterial luciferase to the fluorescent proteins by radiation-less transfer. 

The bacterial bioluminescent reaction requires the contribution of the flavine mononucleotide (FMN) reduced form (FMNH), a long-chain aldehyde (tet-radecanal), and oxygen. All these molecules produce an oxidative reaction in the presence of bacterial luciferase (EC 1.14.14.3), giving tetradecanoic acid, FMN, wateq and hght emission under these conditions. 

The bacterial bioluminescence of Phofobacfer/ t/m phosphoreum results from the interaction of the enzyme luciferase, a flavin mononucleotide in its reduced form, and a long chain (eight carbons or more) aliphatic aldehyde. The presence of oxygen is vital to this interaction. 

Macheroux, P., Eckstein, J., and Ghisla, S., Studies on the mechanism of bacterial bioluminescence. Evidence compatible with a one electron transfer process and a CIEEL mechanism in the luciferase reaction, in Flavins and Flavoproteins, Edmondson, D.E. and McCormick, D.B., Eds., Walter deGruyter, Berlin, 1987, p. 613. 

Holzman, T.F. and Baldwin, T.O., Reversible inhibition of the bacterial luciferase catalyzed bioluminescence reaction by aldehyde substrate kinetic mechanism and Ugand effects. Biochemistry, 22, 2838, 1983. 

Eley, M., j. Lee, J. M. Lhoste, C. Y. Lee, M. J. Cormier, and P. Hemmerich Bacterial Bioluminescence. Comparisons of Bioluminescence Emission Spectra, the Fluorescence of Luciferase Reaction Mixtures, and the Fluorescence of Flavin Cations. Biochemistry 9, 2902 (1970). 

Bioluminescence can also be used as the basis for immunoassay. For example, bacterial luciferase has been used in a co-immobilized system to detect and quantify progesterone using a competitive immunoassay format (34), and other luciferase-based immunoassays have been used to quantify insulin, digoxin, biotin, and other clinically important analytes (35). 

ImmunO lSS iy. Chemiluminescence compounds (eg, acridinium esters and sulfonamides, isoluminol), luciferases (eg, firefly, marine bacterial, Benilla and Varela luciferase), photoproteins (eg, aequorin, Benilld), and components of bioluminescence reactions have been tested as replacements for radioactive labels in both competitive and sandwich-type immunoassays. Acridinium ester labels are used extensively in routine clinical immunoassay analysis designed to detect a wide range of hormones, cancer markers, specific antibodies, specific proteins, and therapeutic dmgs. An acridinium ester label produces a flash of light when it reacts with an alkaline solution of hydrogen peroxide. The detection limit for the label is 0.5 amol. 

Balny, C., and Hastings, J. W. (1975). Fluorescence and bioluminescence of bacterial luciferase intermediates. Biochemistry 14 4719-4723. 

Lee, J., O Kane, D. J., and Gibson, B. G. (1989). Bioluminescence spectral and fluorescence dynamics study of the interaction of lumazine protein with the intermediates of bacterial luciferase bioluminescence. Biochemistry 28 4263-4271. 

Legocki, R. P., Legocki, M., Baldwin, T. O., and Szalay, A. A. (1986). Bioluminescence in soybean root nodules demonstration of a general approach to assay gene expression in vivo by using bacterial luciferase. Proc. Natl. Acad. Sci. USA 83 9080-9084. 

Zeng, J., and Jewsbury, R. A. (1993). Enhanced bioluminescence of bacterial luciferase induced by metal ions and their complexes. In Szalay, A. A., et al. (eds.), Biolumin. Chemilumin., Proc. Int. Symp., 7th, pp. 173-177. Wiley, Chichester, UK. 

Since ideally, a biosensor should be reagentless, that is, should be able to specifically measure the concentration of an analyte without a supply of reactants, attempts to develop such bioluminescence-based optical fibre biosensors were made for the measurements of NADH28 30. For this purpose, the coreactants, FMN and decanal, were entrapped either separately or together in a polymeric matrix placed between the optical fibre surface and the bacterial oxidoreductase-luciferase membrane. In the best configuration, the period of autonomy was 1.5 h during which about twenty reliable assays could be performed. 

W. Huang, A. Feltus, A. Witkowski, and S. Daunert, Homogeneous bioluminescence competitive binding assay for folate based on a coupled glucose-6-phosphate dehydrogenase-bacterial luciferase enzyme system. Anal. Chem. 68, 1646-1650 (1996). 

Chemical immobilization procedures of bioluminescent enzymes such as firefly luciferase and bacterial luciferase-NAD(P)H FMN oxidoreductase to glass beads or rods [174, 175], sepharose particles [176], and cellophane films [177] have produced active immobilized enzymes. Picomole-femtomole amounts of ATP or NAD(P)H could be detected using immobilized firefly luciferase or bacterial luciferase-oxidoreductase, respectively. 

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