Flavin binding by bacterial luciferase: Affinity chromatography of bacterial luciferase

Immobilized FMN covalently attached to Sepharose-6B-hexanoate binds bacterial luciferase. Immobilized flavin is also effective in its reduced form as a substrate in the light emitting reaction catalyzed by luciferase.     

Structure of bacterial luciferase

The generation of light by living organisms such as fireflies, glow-worms, mushrooms, fish, or bacteria growing on decaying materials has been a subject of fascination throughout the ages, partly because it occurs without the need for high temperatures. The chemistry behind the numerous bioluminescent systems is quite varied, and the enzymes that catalyze the reactions, the luciferases, are a large and evolutionarily diverse group. The structure of the best understood of these intriguing enzymes, bacterial luciferase, has recently been determined, allowing discussion of features of the protein in structural terms for the first time.     

Intermediates in the bacterial luciferase reaction

Rapid formation of an unstable, intermediary state of an enzyme complex, which is obligatory in the bacterial luciferase reaction, was observed on aerobic oxidation of the luciferase-FMNH₂ complex. The rate of decay of this intermediate in the absence of aldehyde was measured. The value obtained coincided with that estimated from the decay of the peak intensity of luminescence observed on addition of aldehyde at intervals after mixing the luciferase-FMNH₂ complex with O₂. A sequential mechanism of the reaction of bacterial luciferase is discussed.     

Dynamic fluorescence properties of bacterial luciferase intermediates

Three fluorescent species produced by the reaction of bacterial luciferase from Vibrio harveyi with its substrates have the same dynamic fluorescence properties, namely, a dominant fluorescence decay of lifetime of 10 ns and a rotational correlation time of 100 ns at 2 degrees C. These three species are the metastable intermediate formed with the two substrates FMNH2 and O2, both in its low-fluorescence form and in its high-fluorescence form following light irradiation, and the fluorescent transient formed on including the final substrate tetradecanal. For native luciferase, the rotational correlation time is 62 or 74 ns (2 degrees C) derived from the decay of the anisotropy of the intrinsic fluorescence at 340 nm or the fluorescence of bound 8-anilino-1-naphthalenesulfonic acid (470 nm), respectively. The steady-state anisotropy of the fluorescent intermediates is 0.34, and the fundamental anisotropy from a Perrin plot is 0.385. The high-fluorescence intermediate has a fluorescence maximum at 500 nm, and its emission spectrum is distinct from the bioluminescence spectrum. The fluorescence quantum yield is 0.3 but decreases on dilution with a quadratic dependence on protein concentration. This, and the large value of the rotational correlation time, would be explained by protein complex formation in the fluorescent intermediate states, but no increase in protein molecular weight is observed by gel filtration or ultracentrifugation. The results instead favor a proposal that, in these intermediate states, the luciferase undergoes a conformational change in which its axial ratio increases by 50%.     

A cyanide-aldehyde complex inhibits bacterial luciferase.

Cyanide at high (millimolar) concentrations inhibited in the in vitro Vibrio harveyi luciferase reaction. Cyanide reacted with free aldehyde to form an inhibitor. Inhibitor formation was accelerated by alkaline conditions and bovine serum albumin.     

Bovine serum albumin interacts with bacterial luciferase

Bovine serum albumin (BSA) affects the amount of light obtained from bacterial luciferase by competing with luciferase for one of the luciferase substrates, the aldehyde. At low aldehyde concentrations BSA behaves as an inhibitor, but at high aldehyde concentrations BSA relieves substrate inhibition. BSA reversibly binds decanal with a K_si = 3.36 μmol/l, approximately half the affinity of luciferase for decanal (K_M = 1.5 μmol/l). BSA also increased the rate of intermediate II dark decay. The data suggest that this involves a direct protein-protein (BSA-luciferase) interaction.     

Factors affecting the cellular expression of bacterial luciferase

The in vivo expression of cellular bacterial luciferase has been defined as the luciferase expression quotient, measured as the ratio of the bioluminescence intensity in vivo to the in vitro activity of luciferase in crude cell extracts. The expression is greater in the presence of inhibitors of the electron transport system such as cyanide and N-heptyl-4-hydroxyquinoline and also at lower oxygen tensions. The higher expression of the cellular luciferase under these conditions is postulated to be due to an increase in the intracellular levels of reduced coenzymes which enhance both the reduction of flavin and the reduction of fatty acid to aldehyde. Both FMNH₂ and aldehyde are substrates in the light emitting reaction.     

Photokinetic microanalysis of NADP+, using bacterial luciferase

Summary Bioluminescence photokinetic assay of NADP⁺ is described, using the glucose-6-phosphate dehydrogenase reaction for conversion to its reduced form and subsequent measurement of this with luciferase extracts of Vibria fisherii. The analyses were applied to the determination of the activity of minute amounts of glutathione reductase using NADP⁺ as measurable product and for nucleotide assay in cell samples of 0.5–10 g dry weight. The sensitivity was sufficient for determining 0.5 picomoles NADP⁺.Previously, FMN, NADH, NAD⁺ and NADH have been analysed with the bacterial luciferase system. Its applicability has now been extended by the assay of NADP⁺.     

Thermostability of bacterial luciferase expressed in different microbes

Bacterial luciferase was used to investigate the relationship between the thermostability of a cytoplasmic reporter molecule and cellular heat resistance. The luciferase activity of Vibrio fischeri was expressed in strains of Escherichia coli, Salmonella typhimurium, Listeria monocytogenes and Brochothrix thermosphacta following transformation with plasmid pSP13 carrying the luxAB genes. The thermostability of intracellular luciferase varied depending on the organism in which it was expressed, but was not related to the cellular heat resistance of the different organisms. Addition of xylitol to the heating medium protected against loss of viability and inactivation of intracellular luciferase. Glycerol also protected against loss of viability but was less effective at preventing thermal denaturation of luciferase.     

Activity and subunit functions of immobilized bacterial luciferase

Immobilized luciferase was studied with regard to its reactivity and subunit functions. When immobilized on a matrix (Sepharose 6B), neither the alpha nor the beta subunit alone exhibited luciferase activity. However, for both subunits (so attached), denaturation followed by renaturation in the presence of the second subunit resulted in the recovery of activity on the matrix. It was thus confirmed that both of the two different subunits (alpha and beta) are required for luciferase activity, even after immobilization. Recovery of activity was approximately the same or slightly less with alpha-immobilized luciferase compared with the beta-immobilized enzyme under our experimental conditions. Generally, immobilized luciferase exhibited both a lower FMNH₂ binding affinity and maximum light emission activity in comparison with free native luciferase, but surprisingly, it exhibited no change in the rate constant for the luminescence, this being a measure of the catalytic turnover time. The alpha-subunit-immobilized (renatured with beta) luciferase possessed a lower FMNH₂ binding affinity compared with beta-subunit-immobilized (renatured with alpha) luciferase. Since the protein attachment to the CNBr-activated Sepharose 6B occurs by way of an amino group of luciferase, it was suggested that the binding of FMNH₂ on luciferase, but not the subsequent catalytic steps, is dependent upon some exposed amino groups on both alpha and beta subunits.     

Fluorescence and bioluminescence of bacterial luciferase intermediates.

An intermediate in the luciferase-catalyzed bioluminescent oxidation of FMNH2, isolated and purified by chromatography at -20degrees, was postulated to be an oxygenated reduced flavin-luciferase. Maintained and studied at -20 to -30degrees, this material exhibits a relatively weak fluorescence emission peaking about 505 nm when excited at 370 nm. It may comprise more than one species. Upon continued exposure to light at 370 nm, the intensity of this fluorescence increases, often by a factor of 5 or more, and its emission spectrum is blue shifted to a maximum at about 485 nm. Upon warming its fluorescence is lost and the fluorescence of flaving mononucleotide appears. If warming is carried out in the presence of a long chain aldehyde, bioluminescence occurs, with the appearance of a similar amount of flavine fluorescence. The bioluminescence yield is about the same with irradiated and nonirradiated samples. The bioluminescence emission spectrum corresponds exactly to the fluorescence emission spectrum of the intermediate formed by irradiation, implicating the latter as being structurally close to the emitting species in bioluminescence.     

Single bacterial cell detection using a mutant luciferase

Previously, we constructed a genetically modified luciferase of Photinus pyralis that generates more than 10-fold higher luminescence intensity than the wild-type enzyme. In this study, we demonstrate that this modified luciferase enables us to detect ATP at 10⁻¹⁸ mol, which is almost equal to the quantity contained in a single bacterial cell. Consequently,we have been able to detect bacterial contamination in samples as low as one colony-forming unit (c.f.u.) for both Escherichia coli and Bacillus subtilis.     

Mechanism of inhibition of bacterial luciferase by anaesthetics.

The effects of volatile anaesthetics on bacterial luciferase were studied $\text{in vitro}$. It was shown that the concentration of anaesthetic required to inhibit the reaction velocity by 50% was similar to that required to reduce light output by 50% $\text{in vivo}$ and this concentration was also in the clinical range for each agent. A kinetic response suggestive of competitive inhibition is occuring at the aldehyde binding site on the luciferase and it is postulated that this is related to the very hydrophobic nature of this site.     

Action of phenobarbital on bacterial luciferase of Photobacterium fischeri

The effect of phenobarbital, a typical substrate of monooxygenases from higher organisms, on bioluminescence of the marine bacterium Photobacterium fischeri and bacterial luciferase was studied. Phenobarbital was shown to be an effective quenching agent owing to the interaction with cytochrome P-450, a terminal luciferase component. A competitive interrelation was found between phenobarbital and an aliphatic aldehyde, the substrate of the luminescent reaction.     

Separation of a blue fluorescence protein from bacterial luciferase

Luciferase preparations from two species of marine bioluminescent bacteria, $\text{Photobacterium phosphoreum}$ and $\text{Photobacterium fischeri}$, are shown to contain a low molecular weight protein, containing a blue fluorescence chromophore having an emission maximum in the 470 nm region. A procedure for separating the luciferase and purifying this protein is described. On disc gel electrophoresis the bulk of the protein is observed to migrate along with the blue fluorescence.     

Use of bacterial luciferase for determining monoamine oxidase activity

A bioluminescence assay is proposed for measuring monoamine oxidase activity in different biological specimens (platelets, mitochondria). The assay is based on the bioluminescent reaction catalysed by bacterial luciferase and coupled to monoamine oxidase. Two modifications of the bioluminescence assay were used. In the first case, the bioluminescent system was added to monoamine oxidase preincubated with the substrates, while in the second case, all the components of the coupled enzymatic systems were directly mixed in a cell. The proposed bioluminescence assay is simple, highly sensitive and rapid, and could be especially useful for biomedical examinations.