Accepted Article

Received Date : 30-Dec-2014 Accepted Date : 03-Feb-2015 Article type

Luciferin

: Research Article

Regenerating

Enzyme

mediates

firefly

luciferase

activation through direct effects of D-cysteine on luciferase structure and activity

Roohullah Hemmati1, Saman Hosseinkhani*1, Reza H. Sajedi1, Taha Azad1, Amin Tashakor1,

Nuredin Bakhtiari1, and Farangis Ataei1 1. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.

*

Corresponding author: Saman Hosseinkhani.

Tel: 98 21 8288 4407 Fax: 98 21 8288 4457 Email: [email protected]

Abstract Luciferin regenerating enzyme (LRE) contributes to in vitro recycling of D-luciferin. In this paper, reinvestigation of the luciferase- based LRE assay is reported. Here, using quick change sitedirected mutagenesis seven T-LRE (Lampyris turkestanicus LRE) mutants were constructed and the most

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functional mutant of T-LRE (T69R) was selected for this research and the effects of D- and L-cysteine on T69R T-LRE-luciferase coupled assay are examined. Our results demonstrate that bioluminescent signal of T69R T-LRE-luciferase coupled assay increases and then reach equilibrium state in the presence of 5

mM D-cysteine. In addition, results reveal that 5 mM D- and L-cysteine in the absence of T69R T-LRE

cause a significant increase in bioluminescence intensity of luciferase over a long time as well as decrease in decay rate. Based on activity measurements, far-UV CD analysis, ANS fluorescence and DLS (Dynamic light scattering) results, D-cysteine increases the activity of luciferase due to weak redox potential, anti-aggregatory effects, induction of changes in conformational structure and kinetics properties. In conclusion, in spite of previous reports on the effect of LRE on luciferase bioluminescent intensity, the majority of increase in luciferase light output and time-course originate from the direct effects of D-cysteine on structure and activity of firefly luciferase.

Abbreviations LRE

T-LRE CD Far-UV CD IPTG T69R T-LRE ANS DLS ATP AMP L-AMP [14C]HBT CHBT CoA BME DTT Ni-NTA PCR

Luciferin regenerating enzyme Lampyris turkestanicus LRE Circular dichroism Far-ultra violet circular dichroism Isopropyl-D-thiogalactopyranoside A single mutant of T-LRE in which Threonine 69 replaced by Arginine 1-anilino-8-naphthalene sulfonate Dynamic light scattering Adenosine triphosphate Adenosine monophosphate Dehydroluciferyladenylate 2-[14C] cyano-6-hydroxybenzothiazole 2-cyano-6-hydroxybenzothiazole Coenzyme A Beta-mercaptoethanol Dithitreitol Ni2+-nitrilotriacetate Polymerase chain reaction

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and colleagues have demonstrated the antioxidant properties of cysteine, as a shield to protect enzymes against radiation [24].

In the case of luciferin, based on previous reports, spontaneous condensation of L-cysteine with CHBT results in the production of L-luciferin, and then in the presence of CoA stereoisomeric bio-inversion of L-luciferin to D-luciferin is possible via thioesterase [25-27] . Other studies have provided important evidence on the role of D-cysteine in recycling of D-luciferin in vitro [18-20]. However, the role of LRE in vivo has not been examined as yet. As demonstrated by earlier studies, D-cysteine is added to the luciferase-LRE coupled reaction to recycle D-Luciferin from oxyluciferin [18-20] and newly recycled Dluciferin molecules are consumed by luciferase to intensify the luminescent signal, although Lembert declared that L-luciferin can be used by luciferase as well and managed to produce a weak signal [28].

In this study, we tried to reinvestigate the effect of LRE on luciferase activity in the presence and absence of cysteine. In other words, due to the importance of LRE, at first, this study tries to find unknown aspects of the in vitro luciferase-based LRE assays in the presence of D- and L-cysteine in detail. Secondly, this research explores the influences of D- and L-cysteine on the pattern of light production and the tertiary structure of firefly luciferase in the absence of LRE.

For this, we constructed and screened seven mutants of T-LRE and the most functional single mutant

T69R (Threonine 69 of T-LRE replaced by Arginine) was selected for further investigation of the role of LRE in lucifearse-based assay [29]. The characteristics of this mutant included a high bioluminescent signal in in vitro luciferin-luciferase reaction and a low bioluminescent decay rate over time compared to a wild-type and other mutants of T-LRE [29]. In summary, this study paves the way to enrich our knowledge on the role of LRE and is a step toward clarification and better understanding of the role of LRE and D-cysteine in luciferase–based reactions.

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Materials and methods Reagents Reagents were used in this research are as following: restriction endonuclease DpnI (Fermentas), Kanamycin and Isopropyl-D-thiogalactopyranoside (IPTG) (Sigma), ATP (Roche), D-luciferin potassium salt (ResemBV, the Netherlands), L- and D-cysteine (Sigma-Aldrich), PrimeSTAR GXL DNA Polymerase (Takara Bio), plasmid extraction kit, gel purification and PCR purification kit (GeneAll Biotechnology), Ni-NTA (Ni2+-nitrilotriacetate) spin kit (Novagen Inc), oligonuleotides (Macrogen, Korea),anti His-tag antibodies (Abcam), expressed and purified recombinant luciferase obtained from Iranian firefly Lampyris turkestanicus .

Mutant selection Our results of T-LRE mutants-luciferase coupled assay showed that, T69R single mutant was able to

improve bioluminescence of luciferase more than other mutants and a wild-type T-LRE over the time. T69R single mutant of T-LRE efficiently improved luciferase light emission pattern compared with the wild-type and other mutants of T-LRE. Therefore this single mutant was selected for further investigation of the role of T-LRE in increasing the luciferase activity.

Effects of T69R T-LRE, D-cysteine, L-cysteine and Coenzyme A (CoA) on luciferase

bioluminescence In order to study the effect of T69R T-LRE with and without D-and L-cysteine on light production of

luciferase in comparison with CoA, 20 µl of each compound (50 mM) was added to 180 µl reaction mixture including 0.02 µg luciferase, 0.25 mM D-luciferin, 2 mM ATP, 5 mM MgSO4, 25 mM Tris-HCl buffer (pH 7.8) 15 seconds after the initiation of reaction and the signal variations of luciferase were read by luminometer during 20 minutes at 25 °C. To investigate the effects of T69R T-LRE, 5 µl (2.5 mg.ml -1)

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D- and L-cysteine reactivate heat-inactivated luciferase more effective than DTT, CoA and BME The reactivation of 53 °C heat-inactivated luciferase in the presence of D-cysteine, L-cysteine, DTT, CoA

and BME revealed that L-cysteine and D-cysteine caused a recovery of initial luciferase activity up to 35 % and 25 %, respectively (see supplementary material Figure 3S). Unexpectedly, DTT, CoA, and BME reactivate inactivated luciferase less than L-cysteine and D-cysteine over a short time (see supplementary material Figure 2S).

D- and L-cysteine change the size of heat- aggregated luciferase molecules °

Upon the addition of D- and L-cysteine to a reaction containing heat-inactivated luciferase at 53 C, DLS

data revealed a slight decrease in the number of aggregated molecules without significant change in their particle sizes (see supplementary material Figures 3S and 4S). In contrast to 53 °C, DLS data revealed °

that L-cysteine reduced sizes of 25 C heat- aggregated luciferase molecules more effectively than Dcysteine (Figure 3). The two amino acids were able to decrease the size of aggregations provided upon the ° ° incubation at 25 C more effective than a luciferase sample incubated at 53 C.

Structural changes of luciferase upon the addition of D- and L-cysteine The effects of D/L-cysteine on luciferase structure were studied using intrinsic fluorescence. The emission spectra revealed a decrease in the intensity of intrinsic fluorescence in the presence of Dcysteine and L-cysteine (Figure 4). To study the changes on the surface of firefly luciferase, ANS added to a mixture containing luciferase and either L-cysteine or D-cysteine and as a result a decrease in fluorescence intensity accompanied by a blue-shift in emission peak was observed (Figure 5).

Finally, in order to check the validity of the fluorescence data concerning the luciferase structural change, a far-UV circular dichroism experiment of luciferase accompanied by D-cysteine in comparison with luciferase in the absence of D-cysteine was carried out (Figure 6). Considering the circular dichroism

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θ 208nm and an increase in the value of θ 222nm were observed. Regular

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data, a decrease in the value of

secondary structure estimated by CD software and showed a decrease in beta structures of luciferase in the presence of D-cysteine.

Discussion With recent interest in luciferase -LRE coupled assay; the aim of this research was reinvestigation of the effects of LRE on luciferase light emission in the presence and absence of D-cysteine and L-cysteine. Up to now, interpretations of the results of Luciferase-based LRE assays have not been clearly presented and the influences of D-cysteine on light emission have not been investigated in detail. In this paper, the effects of D- and L-cysteine on the luciferase-based LRE assay have been fully examined. Also, the changes in the activity of firefly luciferase have been studied and reported in the absence and presence of T69R T -LRE with and without L-cysteine and D-cysteine. Moreover, the structural changes of luciferase

have been investigated after the addition of D- and L-cysteine. We also explored the possible effects of Dcysteine, as a component of LRE assay, and L-cysteine on luciferase light production in comparison to CoA. Ultimately, we tried to obtain more information about lucifearse- LRE coupled assay, to provide new insights into unknown aspects of the role of LRE.

Based on results (Figure 1A), the initial rapid increase in the light emission upon the addition of T69R T-

LRE and D-cysteine can be attributed to removal of oxyluciferin by T69R T-LRE and the subsequent constant level of bioluminescence may be correlated with the reaction progress toward an equilibrium state. It seems that CHBT is generated from oxyluciferin at the same rate as it is converted to D-luciferin during the equilibrium state. Obviously, T69R T-LRE exerts a stronger positive effect on bioluminescence over time in the presence of D-cysteine than L-cysteine (Figures 1A and 2A). In the presence of Lcysteine, a smaller increase in the bioluminescence signal and no equilibrium state was observed (Figures

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earlier mentioned results increase in Vmax, induced by D-cysteine, owing to changes in the structure of luciferase can be attributed to a rise in the population of active luciferase molecules or more accessibility of substrates. Thus, the enzyme may be allowed for a greater catalytic efficiency. In other words, it is likely that cysteine reactivated the completely or partially inactivated population of alternative enzyme molecules, giving rise to increase in total active enzyme concentration and increase in Vmax of luciferase

(see Table 1).

Based on the obtained emission spectra, L-cysteine decreased the intrinsic fluorescence emission more

than D-cysteine (Figure 4). This finding is compatible with previous data on the effect of D- and Lcysteine on quenching of fluorescence spectra [36-38]. Along these lines, some researchers have suggested that the flexible site of tryptophan residue is a possible reason for the decrease in intrinsic fluorescence emission intensity without any peak shift [39].

In order to study the possible structural changes of luciferase upon the addition of these two amino acids, an ANS fluorescence experiment was carried out. ANS fluorescence results indicate the decrease in hydrophobic patches at the surface of luciferase (Figure 5). As mentioned above, these amino acids have anti-aggregatory effects on luciferase aggregations, thus, a decrease in ANS fluorescence emission intensity may be due to the removal of hydrophobic patches on the surface of the aggregated lucfierase molecules.

Decrease in the ANS-binding spectra upon addition of the two amino acids is also consistent with that of intrinsic fluorescence. To sum up, a blue-shift together with a reduced intensity of ANS fluorescence in the presence of the two amino acids can be attributed to decrease in hydrophobic pockets at the surface of luciferase and burial of hydrophobic patches in deeper regions within the protein as a result of structural change. Therefore, both ANS and intrinsic fluorescence may indicate local structural changes at the surface of luciferase and in the environment around tryptophan residues.

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The far-UV CD results (Figure 6) showed an induction of alpha helical and a decrease in beta pleated structures. A new conformation for luciferase in the presence of D-cysteine can be deduced from the farUV CD analysis. According to these spectra, it can be concluded that this amino acid might result in stabilization of an intermediate structure of luciferase with improved properties. However, further studies are required to establish the validity of our conjecture about stabilization of an intermediate structure of luciferase after the addition of D-cysteine. Regular secondary structure estimation by CD software revealed a decrease in beta sheet structure and an increase in alpha helix contents. The estimated data

obtained from far-UV CD spectra demonstrate decrease in beta sheet content which may reflect the antiaggregatory effects of D-cysteine.

As mentioned above, ANS fluorescence analysis implies change in the structure of luciferase upon the addition of the two sulfur-containing amino acids. These results are consistent with that of far-UV CD showing a global change in the structure of luciferase. In the other words, global and local structural changes of luciferase in the presence of the two thiol-containing amino acids can be shown by far-UV CD and fluorescence analysis.

Considering the surprising effects of cysteine on luciferase activity over time, it is also possible to suggest that in addition to induction of structural change, cysteine may also remove some inhibitory factors during the luciferase reaction or may gradually be consumed during reaction, due to weak redox potential, to rescue luciferase from oxidation. Increase in luciferase bioluminescence level upon the addition of D-

cysteine in the absence of T69R T-LRE during time was greater than that of in the presence of T69R T-

LRE.

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200 to 250 nm (25 °C) and the background was corrected against a buffer and D-cysteine blank and molar ellipticity determined as reported earlier [3].

Fluorescence measurements Intrinsic fluorescence was measured by exciting the 30 µg. ml-1 enzyme solutions (dissolved in 50 mM

Tris buffer pH 7.8) in the absence and presence of 5 mM D-cysteine or L-cysteine at 295 nm, emission spectra were recorded in the wavelength ranged from 300 to 450 nm at 25 ◦C. Extrinsic fluorescence was

studied using 30 µM 1 ANS as a hydrophobic fluorescent probe. ANS added to a mixture containing 1 µM luciferase (0.062 µg), 5 mM D-cysteine or 5 mM L-cysteine incubated for 5 min at 25 ◦C, followingly

the changes in ANS fluorescence spectra were recorded at the excitation wavelength of 350 nm and the emission wavelength from 400–600 nm. The fluorescence analyzes were conducted by Cary-Eclipse luminescence spectrophotometer (Varian).

Study of the effects of D- and L-cysteine on luciferase aggregation size by DLS The average particle size and the polydispersity of the aggregate-size distribution of the 1µM L.turkestanicus luciferase dissolved in Tris-HCl buffer in presence and absence of D- or L-cysteine at ° ° different temperatures (53 C and 25 C) were determined by DLS using photon correlation ° spectroscopy. The measurements were performed at 25 C and pH 7.8 using a Zetasizer Nano ZS

instrument (Malvern Instruments Ltd, Malvern, Worcestershire, UK) equipped with a helium-neon laser and a scattering angle of 173°. A typical protein refractive index of 1.45 was used. A 50 µl luciferase° ° containing tube incubated at 25 C or 53 C for 5 min added to 950 µl Tris-HCl buffers in the absence

and presence of 5 mM D-cysteine and L-cysteine, then incubated at room temperature for 1 min and the measurements were done.

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Results The effects of T69R T-LRE on the pattern of firefly luciferase light production in the absence and

presence of D- and L-cysteine As shown in Figure 1A, T69R T-LRE increases the light intensity of luciferase over a 20 min period in the

presence of D-cysteine in comparison with the control and L-cysteine-containing reactions. Furthermore, upon the addition of D-cysteine and T69R T-LRE 45% of the initial bioluminescence signal remains after 20 minutes (Figure 1B). In the presence of D-cysteine T69R T-LRE-luciferase-coupled reaction

bioluminescent intensity indicates significant increase compared with that of the L-cysteine (Figures 1A ). On the other hand, T69R T-LRE causes the lowest level of initial increase in bioluminescence in the absence of cysteine; light emission drops to almost zero after 11 min (Figure 1 A). Luciferase assays were performed in the presence of sulfur-containing compounds without T69R T-LRE.

The results indicated that L-cysteine and D-cysteine in the absence of T69R T-LRE enhanced the luciferase activity more than 8- and 7-fold, respectively (Figure 1B). The light emission profile of luciferase was also affected by addition of these amino acids in the absence of T69R T-LRE in such a way

that after 20 min about 44 % of the initial signal remained (Figure 1B).

Here, the shape of curves in figure 1 is seem to be interesting, after 10 seconds, some of inhibitory product and by-product of luciferase are generated in environment in addition to some luciferase molecules aggregants,causing luciferase signal reaching zero. After addition of thiol-containing cysteine during the initial times, a rapid rise in bioluminescent signal is seen which can be due to the ،anti-

oxidative and anti-inhibitory effects and cysteine-induced structural changes on luciferase. In the presence of T-LRE the rapid initial increase in light production may also be correlated with the elimination of oxyluciferin and its conversion to CHBT. In other words a synergistic effects of cysteine on luciferase activity is seen. Over time, the curves reach a stabilized state which may be due to formation of an equilibrium state. The equilibrium state can be formed when a velocity of luciferase reaction is the same

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as T-LRE reaction and condensation of D-cysteine with T-LRE product. Thereby, in the presence of luciferase, T-LRE and D-cysteine a closed cycle is formed that is the conversion of luciferase product to CHBT by T-LRE and its incessant condensation with D-cysteine to recycling of D-luciferin.

Effects of different sulfur-containing compounds such as D-cysteine, L-cysteine and CoA on luciferase light emission In order to find a possible link among the effects of different sulfur-containing compounds on the bioluminescence level and time-course of luciferase,addition of D-cysteine, L-cysteine, and CoA were carried out (Figure 1B). Results revealed that D- and L-cysteine increased the luciferase bioluminescence more than the other mentioned compounds over longer periods; however T69R T-LRE in the presence of

D-cysteine and CoA increased the light intensity more than other compounds, only over the first 3 minutes of the reaction (Figures 1A and 1B). Based on our findings CoA increased the light production more than 15-fold over a short time (Figure 1B); however, 20 min after addition of CoA, about 9 % of the initial bioluminescence signal remained.

Although in the presence of T-LRE the rapid initial increase in light production may also be correlated with the elimination of oxyluciferin and its conversion to CHBT. In other words a synergistic effects of cysteine on luciferase activity is seen. Over time, the curves reach a stabilized state which is possibly due to formation of an equilibrium state an equilibrium state. The equilibrium state can be formed when a velocity of luciferase reaction is the same as those of T-LRE reaction and spontaneous condensation of Dcysteine with T-LRE product or CHBT. Thereby, in the presence of luciferase, T-LRE and D-cysteine a closed cycle is formed that include the conversion of luciferase product (oxyluciferin) to CHBT by TLRE and spontaneous condensation of CHBT with D-cysteine to recycling of D-luciferin.

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Change in decay rate of luciferase bioluminescence in the presence of T69R T-LRE, D-cysteine, Lcysteine and CoA The decay rate of luciferase bioluminescence showed a considerable reduction in the presence of T69R TLRE with or without cysteine (Figure 2A). Compared with L-cysteine, the control and T69R T-LRE without cysteine, simultaneous addition of D-cysteine and T69R T-LRE gave rise to a larger decrease in the luciferase decay rate (Figure 2A). Meanwhile, the light emission of the control reaction reached zero and adding L-cysteine in the presence of T69R T-LRE was more effective than T69R T-LRE without

cysteine and the control reactions increased the light production and the gradual decay of bioluminescence was observed over time (Figure 2A). Moreover, upon the addition of CoA, bioluminescence decay of luciferase increased in comparison with those of D/L-cysteine (Figure 2B).

In figure 2 cysteine is added before bioluminescence initiation, but due to all of above-mentioned over the effects of cysteine on luciferase light production level in figure 1, bioluminescence gradually decreases from zero time to 20 minutes compared with control. In other words, in figure 2 presence of cysteine at zero time prevents bioluminescence reaction to reach zero but by elapsing the time some inhibitory products and oxidants are produced and gradually decrease the level of bioluminescence

Purified and intracellular luciferase light emission changes in the presence of different concentrations of D- and L-cysteine Here, different concentrations of L-cysteine and D-cysteine initially added to luciferase reaction to find the most effective amino acid. By adding up to 15 mM L-cysteine and D-cysteine, during 5 min, the light production of purified recombinant luciferase relatively increased (see supplementary material Figures 1SA and 1SB). At concentrations more than 15 mM, a rapid decrease was observed following the initial increase in luciferase bioluminescence.

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Kinetic parameters of purified luciferase changes in the presence of 5 mM concentrations of D- and L-cysteine Due to bioluminescence enhancement in the presence of D- and L-cysteine, kinetic properties of luciferase with and without cysteine were determined by peak light emissions measurements as relative light units per second (RLU/s). Results revealed that upon the addition of 5 mM of D-cysteine and Lcysteine on luciferase, Km for D-luciferin and ATP have been changed. A decrease in Km and an increase

in Vmax for both substrates of firefly luciferase in the presence of each D- and L-cysteine were observed (Table 1). Moreover, compared with what estimated in the absence of D- and L-cysteine, significant increase in Vmax/Km ratio was calculated in the presence of these two amino acids. In contrast to D-

cysteine, a greater increase was recorded for Vmax/Km ratio in the case of L-cysteine.

Since the effects of L-cysteine are sometimes similar or even higher than that of D-cysteine, it seems that a general mechanism is responsible for this, which has to be elucidated in future.

Table 1. Km,Vmax and Vmax/Km values of luciferase in the absence and presence of 5 mM Lcysteine (L-Cys) and D-cysteine (D-Cys) for both substrates of D-luciferin (LH2) and ATPestimated from Lineweaver–Burk plot. Km Vmax*10 6 Vmax/Km*10 6 (µM) (RLU/s) (RLU.s-1.µM-1) LH2 without Cys LH2 with D-Cys

20 14.2

0.5 1.3

0.025 0.09

LH2 with L-Cys

16.7

3.3

0.2

ATP without Cys

83

0.2

0.002

ATP with D-Cys

61

1.1

0.018

ATP with L-Cys

70

1

0.014

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D- and L-cysteine reactivate heat-inactivated luciferase more effective than DTT, CoA and BME The reactivation of 53 °C heat-inactivated luciferase in the presence of D-cysteine, L-cysteine, DTT, CoA

and BME revealed that L-cysteine and D-cysteine caused a recovery of initial luciferase activity up to 35 % and 25 %, respectively (see supplementary material Figure 3S). Unexpectedly, DTT, CoA, and BME reactivate inactivated luciferase less than L-cysteine and D-cysteine over a short time (see supplementary material Figure 2S).

D- and L-cysteine change the size of heat- aggregated luciferase molecules °

Upon the addition of D- and L-cysteine to a reaction containing heat-inactivated luciferase at 53 C, DLS

data revealed a slight decrease in the number of aggregated molecules without significant change in their particle sizes (see supplementary material Figures 3S and 4S). In contrast to 53 °C, DLS data revealed °

that L-cysteine reduced sizes of 25 C heat- aggregated luciferase molecules more effectively than Dcysteine (Figure 3). The two amino acids were able to decrease the size of aggregations provided upon the ° ° incubation at 25 C more effective than a luciferase sample incubated at 53 C.

Structural changes of luciferase upon the addition of D- and L-cysteine The effects of D/L-cysteine on luciferase structure were studied using intrinsic fluorescence. The emission spectra revealed a decrease in the intensity of intrinsic fluorescence in the presence of Dcysteine and L-cysteine (Figure 4). To study the changes on the surface of firefly luciferase, ANS added to a mixture containing luciferase and either L-cysteine or D-cysteine and as a result a decrease in fluorescence intensity accompanied by a blue-shift in emission peak was observed (Figure 5).

Finally, in order to check the validity of the fluorescence data concerning the luciferase structural change, a far-UV circular dichroism experiment of luciferase accompanied by D-cysteine in comparison with luciferase in the absence of D-cysteine was carried out (Figure 6). Considering the circular dichroism

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θ 208nm and an increase in the value of θ 222nm were observed. Regular

Accepted Article

data, a decrease in the value of

secondary structure estimated by CD software and showed a decrease in beta structures of luciferase in the presence of D-cysteine.

Discussion With recent interest in luciferase -LRE coupled assay; the aim of this research was reinvestigation of the effects of LRE on luciferase light emission in the presence and absence of D-cysteine and L-cysteine. Up to now, interpretations of the results of Luciferase-based LRE assays have not been clearly presented and the influences of D-cysteine on light emission have not been investigated in detail. In this paper, the effects of D- and L-cysteine on the luciferase-based LRE assay have been fully examined. Also, the changes in the activity of firefly luciferase have been studied and reported in the absence and presence of T69R T -LRE with and without L-cysteine and D-cysteine. Moreover, the structural changes of luciferase

have been investigated after the addition of D- and L-cysteine. We also explored the possible effects of Dcysteine, as a component of LRE assay, and L-cysteine on luciferase light production in comparison to CoA. Ultimately, we tried to obtain more information about lucifearse- LRE coupled assay, to provide new insights into unknown aspects of the role of LRE.

Based on results (Figure 1A), the initial rapid increase in the light emission upon the addition of T69R T-

LRE and D-cysteine can be attributed to removal of oxyluciferin by T69R T-LRE and the subsequent constant level of bioluminescence may be correlated with the reaction progress toward an equilibrium state. It seems that CHBT is generated from oxyluciferin at the same rate as it is converted to D-luciferin during the equilibrium state. Obviously, T69R T-LRE exerts a stronger positive effect on bioluminescence over time in the presence of D-cysteine than L-cysteine (Figures 1A and 2A). In the presence of Lcysteine, a smaller increase in the bioluminescence signal and no equilibrium state was observed (Figures

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1A and 2A). Reportedly, L-luciferin inhibits the light emission of luciferase [28, 31], thereby the addition of L-cysteine leads to a gradual inhibition of luciferase over time, which can be attributed to production of L-luciferin from L-cysteine and CHBT in the T69R T-LRE coupled reaction (Figures 1A and 2A).

Furthermore, in the absence of D- and L-cysteine, no equilibrium state was observed during luciferaseT69R T-LRE coupled assay (Figure 1A). This revealed that the rapid initial increase in light production may be correlated with the elimination of oxyluciferin and its conversion to CHBT. Moreover, the results demonstrated that the co-existence of D-cysteine and T69R T-LRE were crucially important for the

formation of equilibrium state (Figure 1A). Interestingly, when luciferase reaction was coupled with heatinactivated T69R T-LRE, neither an equilibrium state nor an increase in light amount was observed in the

absence of both L-cysteine and D-cysteine. It is noteworthy that the formation of equilibrium state observed in this study has not been previously reported [18, 19].

One unanticipated finding in this experiment was the patterns of initial rising in bioluminescent signal for T69R T-LRE, T69R T-LRE with L-cysteine, and T69R T-LRE with D-cysteine in the first 3 minutes,

similar to that of CoA (Figures 1A and 1B). It has been shown in previous studies that CoA eliminates the inhibitory effect of L-AMP [16, 21] and results in a rapid initial increase in bioluminescence intensity. Moreover, it is believed that the inhibitory effects of L-AMP are stronger than oxyluciferin [15], and thus a higher rapid initial increase in light production occurs upon the addition of CoA. Based on our results, considering the removal of the weaker inhibitory effect of oxyluciferin via T69R T-LRE, the rapid initial

increase in levels of light production in the presence and absence of D- and L-cysteine are still lower than CoA, which is consistent with previous remarks (1A and 1B) [16, 21].

In addition, as shown in Figure 2A, the rate of luciferase bioluminescence decay showed a considerable decrease in the presence of T69R T-LRE with or without cysteine which can be attributed to removal of oxyluciferin as an inhibitor of luciferase. Adding D-cysteine to luciferase-based LRE assay leads to a

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greater decrease in luciferase decay rate; somehow, after 20 min more than 25 % of the initial luciferase bioluminescence signal remains (Figure 2A). It can be attributed to removal of a luciferase inhibitor, oxyluciferin, by T69R T-LRE and regeneration of D-luciferin in the presence of D-cysteine and it’s

consumption by luciferase.

A problem with the effects of D-cysteine in the absence of T69R T-LRE is that it is more effective on

bioluminescence of luciferase compared with the coexistence of D-cysteine with T69R T-LRE (Figures 1A, 1B, 2A and 2B). A possible explanation for this might be that an enzyme such as D-cysteine desulfhydrase [32, 33] exists in T69R T-LRE-containing bacterial cell extracts, which removes a number

of D-cysteine amino acids. T69R T-LRE in this study was expressed as a tagged protein to His-tail,but its expression as soluble form was low and we tried many times to purify the active protein using Ni-NTA-Agarose or refold it, but it was not accomplished. The majority of T-LRE was being expressed as inclusion body in bacteria and even application of different approaches to its refolding as active T69R T-LRE was not successful.

However, due to those problems, unpurified T69R T-LRE for this study was used inevitably.

Thus, a possible explanation is consumption of D-cysteine to recycle D-luciferin in the presence of T69R T-LRE. If D-cysteine molecules remain in environment and not be consumed to recycle D-luciferin they will exert stronger effects on luciferase bioluminescence.

The most likely explanation for the deficiency of CoA in luciferase activity retention in comparison with cysteine (Figures 1B and 2B) is that, based on previous reports, coenzyme A acts only as an antiinhibitory compound [16] and therefore can’t improve the luciferase bioluminescence time-course as much as cysteine which may act on luciferase structure or act as anti-oxidant.

It is noteworthy to be mentioned that, upon fortuitous addition of D-cysteine to a cell extract containing

a split luciferase-based biosensor used to detect apoptosome formation [7], bioluminescence intensity decreased to half and then increased [data not shown]. However, after the addition of CoA to the

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mentioned biosensor system, the amount of light production increased. It seems, the effects of CoA and D-cysteine on the biosensor are not similar.

Since, D- and L-cysteine exert vigorous effects on bioluminescence profile (Figures 1B and 2B); we designed a strategy to understand these effects in detail and also investigate the future applications in luciferase assay buffer. At concentrations more than 15 mM, a rapid decrease was observed following the initial increase, which is probably due to inhibitory or quenching effects of these concentrations (see supplementary material Figure 1SB).

Although we assayed T69R T-LRE by a similar protocol, some contradictions exist between our findings

and the previously reported results [18, 19]. For example, it had been reported that the addition of 5 mM D-cysteine to luciferase-LRE reaction recycles oxyluciferin to D-luciferin and declared that light emission of the control reaction extinguishes after 10 min [18]. Contrary to previous reports; our investigation showed that in the presence of 5 mM D-cysteine without T69R T-LRE, 40 % of initial light level was

maintained after 20 min (Figure 2B). Moreover, the addition of 5 mM D-cystein to luciferase reaction in the absence of T69R T-LRE caused an 8-fold increase in luciferase bioluminescence over 20 min (Figure 2B). In our study, the results obtained from the effects of D-cysteine in the presence of LRE on luciferinluciferase reaction are compatible with what had been previously presented, in which coexistence of Dcysteine and T69R T-LRE increased luciferase light production and decreased the bioluminescent decay rate [18, 19].

° The effects of cysteine on luciferase reactivation of 53 C heat-inactivated luciferase in the presence of D-

cysteine, L-cysteine, DTT, BME and CoA were examined. Since the redox potentials of BME and DTT are 0.26 V and 0.33 V [34, 35], respectively, the most likely explanation for more effective effects of Dand L-cysteine on reactivation of heat-inactivated luciferase molecules is that the higher redox potential

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of sulfur-containing compounds leads to a lower reactivation of luciferase (see supplementary material Figure 2S). Hence, D- and L-cysteine can be used for improving the level and the time-course of luciferase bioluminescence during the enzyme assay.

For anti-aggregatory effects of D- and L-cysteine on luciferase molecules, results showed that the two ° amino acids have some effects on 53 C heat-aggregated luciferase molecules (see supplementary

material Figures 2S and 3S). However, DLS data revealed a quite remarkable but unexpected finding °

about the effects of those two amino acids on luciferase molecules incubated at 25 C as L-cysteine

reduced aggregated luciferase size more effectively than D-cysteine (Figure 3). These surprising results showed that possibly D- and L-cysteine due to their weak redox potential reduced a few disulfide bridges, which appeared upon oxidation, among the luciferase aggregations. According to these findings these amino acids may alter the structure of luciferase molecules.

It must also be noted that, in spite of a slight decrease in the sizes of luciferase aggregations, upon ° incubation at 53 C, in the presence of the two amino acids, a higher rate of luciferase reactivation was

observed (Figure 3). That is to say that reactivation of heat-inactivated luciferase by these amino acids does not necessarily indicate the removal of aggregations, but the reactivation of partially or completely unfolded luciferase molecules with a slight change in aggregated molecules population may be the main reason for this issue. Finally, based on the results of the latter experiment, D- and L-cysteine may help reactivation of luciferase through refolding of some partially unfolded protein in solution.

As the values of both Vmax and Km of the reaction changed in the presence of 5 mM L-cysteine and D-

cysteine, it is thought that both the enzyme structure and its binding to the substrate were affected. In fact, in the presence of these two amino acids not only the values of Km decreased and Vmax increased but also Vmax/Km ratio increased compared with what calculated in the absence of D- and L-cysteine. Based on our

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earlier mentioned results increase in Vmax, induced by D-cysteine, owing to changes in the structure of luciferase can be attributed to a rise in the population of active luciferase molecules or more accessibility of substrates. Thus, the enzyme may be allowed for a greater catalytic efficiency. In other words, it is likely that cysteine reactivated the completely or partially inactivated population of alternative enzyme molecules, giving rise to increase in total active enzyme concentration and increase in Vmax of luciferase

(see Table 1).

Based on the obtained emission spectra, L-cysteine decreased the intrinsic fluorescence emission more

than D-cysteine (Figure 4). This finding is compatible with previous data on the effect of D- and Lcysteine on quenching of fluorescence spectra [36-38]. Along these lines, some researchers have suggested that the flexible site of tryptophan residue is a possible reason for the decrease in intrinsic fluorescence emission intensity without any peak shift [39].

In order to study the possible structural changes of luciferase upon the addition of these two amino acids, an ANS fluorescence experiment was carried out. ANS fluorescence results indicate the decrease in hydrophobic patches at the surface of luciferase (Figure 5). As mentioned above, these amino acids have anti-aggregatory effects on luciferase aggregations, thus, a decrease in ANS fluorescence emission intensity may be due to the removal of hydrophobic patches on the surface of the aggregated lucfierase molecules.

Decrease in the ANS-binding spectra upon addition of the two amino acids is also consistent with that of intrinsic fluorescence. To sum up, a blue-shift together with a reduced intensity of ANS fluorescence in the presence of the two amino acids can be attributed to decrease in hydrophobic pockets at the surface of luciferase and burial of hydrophobic patches in deeper regions within the protein as a result of structural change. Therefore, both ANS and intrinsic fluorescence may indicate local structural changes at the surface of luciferase and in the environment around tryptophan residues.

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The far-UV CD results (Figure 6) showed an induction of alpha helical and a decrease in beta pleated structures. A new conformation for luciferase in the presence of D-cysteine can be deduced from the farUV CD analysis. According to these spectra, it can be concluded that this amino acid might result in stabilization of an intermediate structure of luciferase with improved properties. However, further studies are required to establish the validity of our conjecture about stabilization of an intermediate structure of luciferase after the addition of D-cysteine. Regular secondary structure estimation by CD software revealed a decrease in beta sheet structure and an increase in alpha helix contents. The estimated data

obtained from far-UV CD spectra demonstrate decrease in beta sheet content which may reflect the antiaggregatory effects of D-cysteine.

As mentioned above, ANS fluorescence analysis implies change in the structure of luciferase upon the addition of the two sulfur-containing amino acids. These results are consistent with that of far-UV CD showing a global change in the structure of luciferase. In the other words, global and local structural changes of luciferase in the presence of the two thiol-containing amino acids can be shown by far-UV CD and fluorescence analysis.

Considering the surprising effects of cysteine on luciferase activity over time, it is also possible to suggest that in addition to induction of structural change, cysteine may also remove some inhibitory factors during the luciferase reaction or may gradually be consumed during reaction, due to weak redox potential, to rescue luciferase from oxidation. Increase in luciferase bioluminescence level upon the addition of D-

cysteine in the absence of T69R T-LRE during time was greater than that of in the presence of T69R T-

LRE.

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Conclusion According to the results, it can be concluded that regeneration of D-luciferin at saturated concentration used in previous studies [18-20] will not effectively increase the luciferase activity. It also may be deduced that in spite of the previous reports on the direct role of LRE in regeneration of D-luciferin in the presence of D-cysteine and oxyluciferin and increase in luciferase light output, most of this increase occur from the direct effects of D-cysteine on firefly luciferase structure and activity. This conclusion has more been guaranteed with the evaluation of separate addition of both D/L-cysteine to luciferase reaction in the absence of T69R T-LRE. However, LRE remains as a unique enzyme able to recycle D-luciferin in the presence of D-cysteine even at the lowest concentration of D-luciferin in luciferase-LRE coupled reaction.

Acknowledgment This work was supported by a grant from the Iranian National Science Foundation (INSF). Research Council of Tarbiat Modares University is also acknowledged for financial support of this work.

Supplementary materials A supporting information file can be found on Wiley Online Library; DOI: xxxxx

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FIGURE CAPTIONS Fig.1. (A) Effects of T69R T-LRE on luciferase bioluminescence in the presence of D-cysteine (D-Cys+T-LRE)

and L-cysteine (L-Cys+TLRE) and absence of cysteine (T-LRE). (B) Effects of D-cysteine (D-Cys), L-cysteine (LCys) and coenzyme A (CoA) on luciferase bioluminescence. D-Cys+T-LRE, closed diamond; L-Cys+TLRE ,open triangle, T-LRE, open circle ; control, solid line; CoA, closed square; L-Cys, closed triangle; D-Cys, closed diamond.

Fig.2. (A) The effects of T69R T-LRE on decay rate of luciferase bioluminescence in the presence of D-cysteine

(D-Cys+T-LRE) and L-cysteine (L-Cys+T-LRE) and absence of cysteine (T-LRE). (B) effects of D-cysteine (D-

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Cys), L-cysteine (L-Cys), and coenzyme A (CoA) on lucierase bioluminescence decay rate. D-Cys+ T-LRE, closed square; L-Cys+ T-LRE, closed circle; T-LRE, closed diamond; control, solid line; CoA, closed square; L-Cys, closed diamond ; D-Cys, solid line; control, dashed line.

Fig.3. The effect of D- and L-cysteine on aggregation sizes of luciferase molecules. A 50µl (1µM) of luciferase sample incubated at 25 °C for 5 min was added to 950 µl Tris-HCl buffers in the absence (c) and presence of 5 mM L-cysteine (a) and D-cysteine (b). The average aggregate-size and the polydispersity of the aggregate-size distribution were determined by DLS.

Fig.4. Intrinsic fluorescence spectra of luciferase samples in the presence and absence of cysteine. The fluorescence

analysis were conducted by Cary-Eclipse luminescence spectrophotometer (Varian). Luciferase (LUC), closed

square; luciferase together with L-cysteine (LUC+ L-Cys), dashed line; luciferase together with D-cysteine (LUC+ D-Cys), solid line.

Fig.5. Extrinsic fluorescence of luciferase in the presence of 30 µM of ANS as a hydrophobic fluorescent probe in the presence and absence of D- and L-cysteine. ANS was added to a mixture containing 1 µM luciferase (0.062 µg), 5 mM D- or L-cysteine incubated for 5 min at 25 ◦C, then the changes in ANS fluorescence spectra were recorded.

Luciferase (LUC) +ANS, closed square; luciferase together with L-cysteine and ANS (L-Cys +LUC+ANS), solid line; luciferase with D-cysteine and ANS (D-Cys+ LUC+ANS), dashed line.

Fig.6. Far-UV CD spectera of luciferase samples in the presence (dashed line) and absence (solid line) of 5 mM D-

cysteine. All spectra collected from 200 to 250 nm and the background was corrected against a buffer or a Dcysteine blank. Luciferase in the presence of D-Cysteine, dashed line; luciferase in the absence of D-Cysteine, solid line.

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Supporting material Fig.1S. (A) Effects of different concentrations of D-cysteine (D-Cys) and L-cysteine (L-Cys) up to 7.5 mM on luciferase bioluminescent signal (7.5 mM L-Cys, closed square;7.5 mM D-Cys, closeddiamond; 5

mM L-Cys, closed triangle; 5 mM D-Cys, closed circle; 2.5 mM L-Cys, dashed line; 2.5 mM D-Cys, solid line; control, open square). (B) Effects of 15 and 25 mM concentrations of D-cysteine (D-Cys) and L-

cysteine (L-Cys) on luciferase bioluminescence signal. A 20 µl of each concentration of D-Cysor LCyswas added 15 seconds after the initioation of reaction to 180 µl reaction mixture include 0.03 µg (0.03 mg.ml-1) luciferase, 0.25 mM D-luciferin, 1.5 mM ATP, 6 mM MgSO4, 30 mM Tris-HCl buffer (pH 7.8)

and the signal variations of luciferase were recorded by luminometer during 20 minutes at 25 °C. (15 mM L-Cys, closed square;15 mM D-Cys, closeddiamond; 25 mM L-Cys, closed triangle; 25 mM D-Cys, dashed line; control, solid line)

Fig.2S.Effects of D-cysteine (D-Cys), L-cysteine (L-Cys), beta-mercaptoethanol (BME), ditiotreitol (DTT), and coenzyme A (CoA) on reactivation of heat-inactivated luciferase. 20 µl luciferase inactivated at 53 °C for 5 min, cooled on ice for 5 min and subsequently 10 µl (0.2 mg.ml-1) of the

inactivated-cooled enzyme added to 40 µl mixture include 0.25 mM D-luciferin, 1.5 mM ATP, 6 mM MgSO4, 30 mM Tris-HCl buffer (pH 7.8) and 6 mM of each of D-Cys, L-Cys, BME, DTT and CoA. Experiments were carried out in presence of these compounds and rates of reactivation over time were measured with luminometer (Berthold Detection System, Germany) at 25 °C (pH 7.8). Readings were

taken every 20 seconds (CoA, closed triangle; L-Cys, closed diamond;DTT, dashed line; BME, open square;DCys, closed square; control, closed circle).

Fig.3S. The effect of L-cysteine on luciferase aggregation size. 50µl (1µM) of heat-inactivated luciferase sample at 53 °C for 5 min was added to 950 µl Tris-HCl buffers in the absence (a) and presence (b) of 5 mM L-cysteine. The average aggregate size and the polydispersity of the aggregate-size distribution were determined by dynamic light scattering (DLS).

Fig.4S.The effect of D-cysteine on luciferase aggregation size.50µl (1µM) of heat-inactivated ° Luciferase sample at 53 C for 5 min was added to 950 µl Tris-HCl buffers in the absence (a) and

presence (b) of 5 mM D-cysteine. The average aggregate size and the polydispersity of the aggregate-size distribution were determined by dynamic light scattering (DLS).

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Figures

Fig.1

Fig.2

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Fig.3.

Fig.4.

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Fig.5.

Fig.6.

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