Difference between revisions of "Part:BBa K5043010"

Latest revision as of 09:22, 2 October 2024

phtAd from M. vanbaalenii Pyr-1

phtAd codes for phthalate dioxygenase ferredoxin reductase. It is a subunit of various ring-hydroxylating oxygenase enzyme complexes which participate in biodegradation of polycyclic aromatic hydrocarbons. [1–3] This part encodes for the same protein as BBa_J73043. It only differs in synonym codon usage.

Background

It is well-established that phtAc and phtAd together function as an electron transport chain, facilitating electron transfer from NADH [1].This system is crucial for supplying electrons to various dioxygenase systems, which are involved in the reduction of polycyclic aromatic hydrocarbons (PAHs). One such complex with dioxygenase activity is formed by nidA and nidBtogether with phtAc and phtAd, capable of degrading pyrene into cis-4,5-dihydroxy-4,5-dihydropyrene [2].

In this project, our complex of interest is formed by the ring-hydroxylating dioxygenase system, composed by pdoA2 and pdoB2 . Together with phtAcAd, these components aggregate to form a complex that exhibits dioxygenase activity [4–6]. This complex is capable of converting phenanthrene-4-carboxylate, an intermediate product of pyrene degradation, into cis-3,4-dihydroxy-phenanthrene-4-carboxylate, see Figure 1 [5, 6]. Given this reaction’s importance in pyrene degradation, we opted to conduct a more detailed characterization. The components of the electron transport chain, ferredoxin phtAc and ferredoxin reductase phtAd, were produced and purified to elucidate their kinetic parameters. Subsequently, we aimed to analyze the complex formation with pdoA2B2 through HPLC analysis.

Figure 1: Enzymatic reaction of *phtAcAd-pdoA2B2* complex [3].
Proposed schema of a catalytic cycle mediated by pdoA2B2-PhtAcAd. Reaction: A molecule of dioxygen is integrated into polycyclic aromatic hydrocarbons (PAHs) through the transfer of two electrons supplied by NADH, facilitated by the electron transport complex phtAcAd.

Enzyme production and purification

Enzyme coding sequence was cloned into pQE bacterial expression vector with a N-terminal, hexahistidine tag. Proteins were expressed in E. coli BL21 (DE3). Main cultures were incubated at 37°C until an OD600 of 0.5 was reached. Subsequently, cultures were induced with a final concentration of 0.5 mM IPTG and incubated overnight at 30°C. Purification was performed using Immobilized Metal Affinity Chromatography (IMAC). Both production and purification samples were analyzed via SDS-PAGE and Coomassie staining (data not shown). Enzyme concentration was determined using the Bradford Assay [8]. The final protein yield of phtAd was 105.1 µM.

Characterization of ferredoxin reductase phtAd

The characterization of phtAd activity was performed by evaluating its ability to reduce 2,6-dichlorophenolindophenol (DCPIP), which serves as an electron acceptor [1]. In its oxidized form, DCPIP displays a blue color, which transitions to colorless upon reduction, facilitating the evaluation of phtAd activity, see Figure 2. The decrease in absorbance was monitored at 600nm. NADH and FADH were needed as cofactors [1].

Figure 2: Reduction of DCPIP performed with different *phtAd* concentrations.

Method

The optimal reaction time and enzyme concentration for the assay were determined by measuring the decrease in absorbance at 2-minute intervals over a 30-minute period. This approach was necessary due to the uncertainty regarding the concentration at which the reaction would proceed and the duration for which the enzyme could effectively catalyze the reaction before reaching substrate saturation. Following the determination of these parameters, the reaction was assessed using varying concentrations of NADH while maintaining a constant enzyme concentration to determine the kinetic parameters. The reaction was initiated by the addition of a specified amount of phtAd and monitored at 600nm using a UV/Vis spectrometer.

Results

Determination of the optimal enzyme concentration
Initial optical analysis indicates that the assay conducted with the highest concentration of phtAd, 1.25 µM, resulted in the complete reduction of DCPIP, as evidenced by the solution's transition to a fully colorless state (see Figure 3). On the other hand, Figure 4 shows that there is no significant difference in the absorbance measurements between the assays conducted with 1 µM and 1.25 µM phtAd. However, the reaction conducted with 0.5 µM phtAd exhibited a lesser decrease in absorbance over the 30-minute period, indicating that the reaction proceeded at a relatively slow rate.

Figure 3: Reduction of DCPIP with different *phtAd* concentrations [μM].

Regarding the optimal reaction time, Figure 4 illustrates that within the initial 10 minutes, absorbance decreases rapidly, indicating that the reduction of DCPIP occurs predominantly during this period. Beyond the 10-minute mark, absorbance measurements stabilize, suggesting that the reaction has reached a saturation point. This phenomenon may be attributed to the complete reduction of DCPIP or the inability of the reaction to proceed further due to the lack of NADH regeneration.

Figure 4: Reduction of DCPIP performed with different *phtAd* concentrations.
The reduction of DCPIP by *phtAd* was monitored for 30 min at a constant substrate quantity and varying amount of enzyme. The reaction mixture contained 100 mM Tris/HCl (pH 7.5), 100 µM NADH, 30 µM DCPIP, 0.5 µM FAD and 0.5, 1, 1.25 µM *phtAd*. Reaction was performed at room temperature.

Based on the results obtained, we selected a concentration of 1.25 µM phtAd for the subsequent assay, as this concentration demonstrated a complete reduction of DCPIP.

Kinetic parameters of phtAd

For the determination of kinetic parameters the assay was run with different NADH concentrations ranging from 40 µM to 250 µM. Data obtained from these assays was then analyzed using Michaelis-Menten kinetics (see Figure 5) and a Lineweaver-Burk plot (see Figure 6) was constructed to provide a linear representation of enzyme kinetics to determine key parameters such as Vmax and Km.

Figure 5: Michaelis-Menten Curve for *phtAd*.
*The velocity *v = ΔE / (ε × d × Δt)* was calculated utilizing the Lambert-Beer Law with the extinction coefficient of DCPIP (ε600 23 mM-1 x cm–1*) [1]*. A logarithmic trend line was applied as a preliminary approximation.*

Figure 6: Lineweaver-Burk plot.
*Graphs show the fitted linear function and its correlation coefficient.*

The Vmax, Km, kcat, kcat/Km values of ferredoxin reductase phtAd were 0.0011 mM/min, 0.028 mM, 1.12 min^-1 and 39.46 mM^-1 x min^-1, respectively. Notable discrepancies from the values reported in the literature [1] were identified, which can be attributed to variations in enzyme concentration. The reaction was performed using a higher enzyme concentration due to the observed diminished activity of the produced enzymes.

Furthermore, the R² value of 0.4668 indicates that the Lineweaver-Burk plot does not perfectly describe the relationship between 1/[NADH] and 1/V. While the fit is moderate, it suggests that factors like experimental variability, biological complexity, or non-linearity in enzyme behavior might be contributing to deviations from the ideal linear relationship.

Characterization of ferredoxin phtAc coupled to phtAd

The activity of phtAc was assessed using a coupled assay. The increase in absorbance resulting from an electron transfer to cytochrome c (electron acceptor) signifies an interaction between ferredoxin phtAc and ferredoxin reductase phtAd. The increase in absorbance was monitored at 550nm. NADH and FADH are needed as cofactors. [1]

Method

Initial measurements were conducted in accordance with the methodology outlined by Wu et al. (2020) [1]. However, the assay produced inconclusive results, as no variation in absorption was observed over time. This lack of change was attributed to the high concentration of cytochrome c (600 µM) utilized in the initial trials. Consequently, we opted to conduct further assays employing varying concentrations of cytochrome c and NADH to identify the optimal concentrations that would facilitate a measurable increase in absorption. In alignment with the approach taken by Wu et al. (2020) [1], subsequent assays were performed using a phtAd to phtAc ratio of 1:3. The increase in absorbance was measured over a 30-minute period at 2-minute intervals at 550 nm. After the optimal cytochrome c concentration was established, further assays were performed varying the NADH concentration to determine the kinetic parameters.

Results

Determination of the optimal substrate concentration

In the initial assay conducted with 250 µM cytochrome c, no change in absorbance was observed (see Figure 7A). Considering this outcome, the subsequent assay was performed using 100 µM cytochrome c in conjunction with a higher concentration of NADH (200 µM). This combination resulted in a measurable increase in absorbance, demonstrating a linear progression over a duration of 26 minutes (see Figure 7C) indicating the oxidation of cytochrome c. To validate this outcome, additional assays were conducted utilizing 100 µM cytochrome c alongside varying concentrations of NADH, both higher and lower. The assay employing 250 µM NADH also demonstrated a linear increase in absorption (Figure 7B) in the first 26 minutes. Conversely, the assay with 100 µM NADH (See Figure 7D) also exhibited a linear increase in absorption, albeit at a reduced rate. Notably, no saturation point was observed at this concentration, in contrast to the higher concentrations, which indicated saturation occurring approximately 30 minutes into the assay.

Figure 7: Oxidation of cytochrome c catalyzed by *phtAc*, in conjunction with *phtAd*, under varying concentrations of cytochrome c and NADH.
The oxidation of cytochrome c by phtAc coupled with phtAd was monitored for 30 min at a constant enzyme concentration and varying amount of substrate. The reaction mixture contained 100 mM Tris/HCl (pH 7.5), 100 – 250 µM NADH, 100 and 250 µM cyt. c, 0.5 µM FAD, 1 µM phtAd and 3.5 µM phtAc. Reaction was performed at room temperature.

Based on the results obtained, we chose a concentration of 100 µM cytochrome c for the subsequent tests, as this concentration showed a measurable increase in absorbance. For the reaction time, we decided to run further reactions for 34 minutes to confirm the saturation point.

Kinetic parameters of phtAc

For the determination of kinetic parameters the assay was run with different NADH concentrations ranging from 40 µM to 400 µM. Data obtained from these assays was then analyzed using Michaelis-Menten kinetics (see Figure 8) and a Lineweaver-Burk plot (see Figure 9) was constructed to provide a linear representation of the enzyme kinetics to determine key parameters such as Vmax and Km.

Figure 8: Michaelis-Menten curve for phtAc.
The velocity *v = ΔE / (ε × d × Δt)*was calculated utilizing the Lambert-Beer Law with the extinction coefficient of cytochrome C (ε550 21 mM^-1 x cm^–1) [6]. A logarithmic trend line was applied as a preliminary approximation.

Figure 9: Lineweaver-Burk plot.
*Graphs show the fitted linear function and its correlation coefficient.*

The Vmax, Km, kcat, kcat/Km values of the ferredoxin phtAc were 0.00043 mM/min, 0.31 mM, 0.12 1/min and 3.9 1/mM x min, respectively. Significant deviations from the literature values [3] were observed; however, the reaction was conducted with a substantially lower concentration of cytochrome c and elevated concentrations of enzymes, as the produced enzymes exhibited reduced activity.

Moreover, the R² value of 0.5378 derived from the kinetic analysis of phtAc is marginally higher than that of phtAd. This marginally elevated value may indicate a greater reliability of the data or a more consistent enzymatic response across varying NADH concentrations. Nevertheless, the Lineweaver-Burk plot does not accurately characterize the relationship between 1/[NADH] and 1/V. Although the fit is moderate, it implies that factors such as experimental variability, biological complexity, or non-linear enzyme behavior could be responsible for the observed deviations from the expected linear relationship.

Activity assay coupled with HPLC analysis

We successfully characterized and demonstrated the activity of phtAc and phtAd both individually and as a complex. This complex functions as an electron carrier for pdoA2B2 [4–6]. The subsequent step involved conducting an activity assay in conjunction with HPLC analysis with the aim to evaluate the function of pdoA2B2 both independently and in complex with phtAcAd, forming a tetramer.

Method

Initially, the activity assay was conducted, wherein the enzymes were incubated at 30°C for approximately 15 to 20 minutes, both in the presence and absence of NADH to examine the effect of NADH on enzymatic aggregation. Subsequently, phenanthrene-4-carboxylate (P4C) was added to initiate the reaction. The reaction mixture was then incubated for 30 minutes at 30°C, after which the reaction was terminated by the addition of 100% methanol. Following this, HPLC analysis was performed using a Zorbax SB-C18 column, employing an acetonitrile/water gradient at a flow rate of 0.4 ml/min, with the column oven maintained at room temperature. The gradient elution program commenced with an initial mobile phase of 60:40 (v/v) acetonitrile to water, transitioning linearly to 100% acetonitrile over 14 minutes, followed by a return to the initial phase (60:40) after 5 minutes. The total duration for each analysis was 35 minutes. For the negative control, an assay was conducted using denatured enzymes, achieved by heating the enzymes at 95°C for 10 minutes. Peaks were analyzed at 270 nm absorbance.

Results

Initial activity assays were performed solely with pdoA2 and pdoB2 to form a dimer, thereby validating their enzymatic activity and evaluating their aggregation characteristics. Monomers were incubated together for aggregation in the presence and absence of NADH to determine whether NADH, as a cofactor, influences the aggregation process.

The efficacy of the reaction was assessed by measuring the absorbance of phenanthrene-4-carboxylate (P4C) and NADH using HPLC. The area under the resulting peaks corresponding to the levels of P4C and NADH was determined, enabling a comparative analysis of the consumption of P4C and NADH against established standards and between the samples. In this context, phenanthrene-4-carboxylate consumption refers to the extent of P4C degradation, as indicated by a reduction in peak area compared to the standard. In the absence of additional samples with different concentrations, it is not possible to quantify the remaining P4C in the samples. The only conclusion that can be drawn is whether P4C has been degraded, as evidenced by the decrease in the area of the corresponding peak; this applies also to NADH.

Furthermore, to eliminate potential statistical errors in the assessment of our results, we employed triplicate measurements for each sample and calculated their averages. The results are presented in Figure 10. Results indicated that the assays in which NADH was added prior to aggregation exhibited enhanced P4C degradation and, correspondingly, increased NADH consumption compared to the assays where NADH was added after aggregation. However, the observed NADH consumption was relatively low, which may be attributed to the absence of the electron carrier dimer (phtAcAd) in the sample, thereby limiting NADH consumption.

Figure 10: Comparative Analysis of phenanthrene-4-carboxylate (P4C) and NADH Consumption in *pdoA2*:*pdoB2* activity assays.
The degradation of P4C by *pdoA2*:*pdoB2* was monitored for 30 min at a constant substrate and enzyme concentration. The reaction mixture contained 100 mM Tris/HCl (pH 7.5), 25 µM P4C, 1 µM *pdoA2* and 1 µM *pdoB2*. The 100 µM NADH were either added after or before aggregation.

Considering the initial results, the analysis of the tetramer aggregation process and the degradation of phenanthrene-4-carboxylate was conducted by introducing NADH prior to the aggregation process.

Degradation of phenanthrene-4-carboxylate by enzyme complex with dioxygenase activity using NADH as a cofactor

The presence of the functional enzyme complex with dioxygenase activity is anticipated to result in a reduction in the quantity of phenanthrene-4-carboxylate. This reduction can be quantified by determining the decrease in the area of the corresponding peak in comparison to the standard. Given the unknown stoichiometry of this enzyme complex, we conducted assays utilizing two distinct stoichiometric ratios. The first assay employed a 1:1 stoichiometry of all monomers, while the second utilized a ratio of pdoA2¹:pdoB2¹:phtAc³:phtAd¹. This latter stoichiometry was selected based on previous kinetic analyses of phtAc, which were performed with a ratio of three equivalents phtAc to one phtAd for determining kinetic parameters. [3] This ratio was employed in the present assays to assess its influence on the degradation of phenanthrene-4-carboxylate and to determine whether the presence of three equivalents phtAc enhanced the efficiency of the electron transfer process, thereby accelerating the breakdown of phenanthrene-4-carboxylate. However, results showed (see Figure 11) that the breakdown of phenanthrene-4-carboxylat was slightly more efficient with the 1:1 ratio of the monomers; this could suggest that a higher ratio of phtAc does not enhance the electron transfer process. And that the complex with 1:1 ratio of all enzymes aggregates better.

To confirm the aggregation of the enzyme complex and the successful degradation of P4C by this complex, negative controls were prepared. For this, assays were prepared with denatured enzymes with the stoichiometries described above. Results showed (see Figure 11) that the peak area of P4C within these assays was higher in comparison with the assays with the active enzyme complex. The comparison of these results indicates that the enzyme complex was successfully formed and demonstrated the capacity to degrade P4C.

Figure 11: Comparative Analysis of phenanthrene-4-carboxylate (P4C) and NADH Consumption in *pdoA2*:*pdoB2*:*phtAc*:*phtAd* activity assays.
The degradation of P4C by the enzyme complex was monitored for 30 min at a constant substrate and enzyme concentration. The reaction mixture contained 100 mM Tris/HCl (pH 7.5), 200 µM NADH, 40 µM P4C, 1 µM pdoA2, 1 µM pdoB2, 1 or 3 µM and 1 µM phtAd.

Another negative control was conducted by excluding any enzymes from the assay. In comparison to samples containing denatured enzymes, the results showed a reduced peak area in the peak corresponding to phenanthrene-4-carboxylate, suggesting either a diminished quantity of P4C in the sample or its degradation. However, the possibility of cross-reactivity with other components in the assay can be discounted, as such interactions would also manifest in the other negative controls. This observation indicates the presence of an independent source of error. Potential issues may involve the unintentional introduction of enzymes or inadequate substrate concentration. The presence of enzymes in the assay can be corroborated by taking into consideration the amount of NADH; notably, the peak area of NADH in this sample was diminished suggesting the possibility that a reaction has taken place, and the results are more consistent with those obtained from the assay conducted with a 1:3 enzyme ratio (see Figure 11). While numerous errors could have arisen, a systematic issue such as cross-reactivity, which would render the enzyme ineffective, can be dismissed by comparing the sample to those with non-functional enzymes.

Moreover, as anticipated, the samples containing the active enzyme complex demonstrated a decrease in the peak area associated with NADH relative to the standard (see Figure 11). This finding corroborates the functionality of the electron carrier dimer, which collaborates with pdoA2B2 in the degradation of P4C. In contrast, the assay involving denatured enzymes at a 1:1 ratio revealed no consumption of NADH, indicating the absence of any reaction.

However, the results of NADH consumption from the assay involving denatured enzymes, utilizing three equivalents of phtAc in conjunction with a 1:1 ratio of the other enzymes, demonstrate a reduction in NADH consumption, as evidenced by the diminished area of the corresponding peak relative to the standard. This observation indicates the presence of an independent source of error, as the other assay performed with denatured enzymes shows no NADH consumption. Potential issues may include insufficient NADH concentration. If the NADH concentration was low, degradation could have occurred over time, as the activity assays were conducted days prior to the HPLC analysis. The thawing process and exposure of the sample to temperature fluctuations may have contributed to NADH degradation due to its inherent instability. Additionally, the possibility of incomplete denaturation should be considered. If the enzymes were not fully denatured during the assay, residual active enzyme could remain, resulting in NADH consumption. The denaturation conditions may not have been adequate to completely inactivate the enzymes. This is further supported by the observation that P4C consumption was slightly lower in comparison to the sample with denatured enzymes with the 1:1 ratio.

Conclusion

In conclusion, the enzyme complex with dioxygenase activity demonstrated its ability to degrade phenanthrene-4-carboxylate (P4C), with slightly greater efficiency observed in the assay utilizing a 1:1 ratio of monomers compared to the 1:3 ratio of phtAc to pdoA2:pdoB2:phtAd. This suggests that a higher ratio of phtAc does not enhance electron transfer efficiency. Negative controls, including assays with denatured enzymes and no enzymes, confirmed that the active enzyme complex was responsible for P4C degradation, while the reduction in NADH in some denatured enzyme assays points to possible errors, such as NADH degradation or incomplete enzyme denaturation. Further assays with stricter controls are needed to confirm these findings and minimize potential sources of error.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 778
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 778
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 778
Illegal XhoI site found at 1124
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 778
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 778
Illegal NgoMIV site found at 1159
1000
COMPATIBLE WITH RFC[1000]

References

[1] Y. Wu, Y. Xu, and N. Zhou, "A newly defined dioxygenase system from Mycobacterium vanbaalenii PYR-1 endowed with an enhanced activity of dihydroxylation of high-molecular-weight polyaromatic hydrocarbons," Front. Environ. Sci. Eng., vol. 14, no. 1, 2020, doi: 10.1007/s11783-019-1193-5.

[2] Kweon, O., Kim, S., Freeman, J. P., Song, J., Baek, S., & Cerniglia, C. E, "Substrate Specificity and Structural Characteristics of the Novel Rieske Nonheme Iron Aromatic Ring-Hydroxylating Oxygenases NidAB and NidA3B3 from Mycobacterium vanbaalenii PYR-1" mBio, 1(2), 2010, doi: 10.1128/mbio.00135-10

[3] S.-J. Kim, O. Kweon, R. C. Jones, J. P. Freeman, R. D. Edmondson, and C. E. Cerniglia, "Complete and integrated pyrene degradation pathway in Mycobacterium vanbaalenii PYR-1 based on systems biology," Journal of bacteriology, vol. 189, no. 2, pp. 464–472, 2007, doi: 10.1128/JB.01310-06.
[4] L. Guo et al., "Characterization of a novel aromatic ring-hydroxylating oxygenase, NarA2B2, from thermophilic Hydrogenibacillus sp. strain N12," Applied and environmental microbiology, vol. 89, no. 10, e0086523, 2023, doi: 10.1128/aem.00865-23.
[5] C. Pagnout, G. Frache, P. Poupin, B. Maunit, J.-F. Muller, and J.-F. Férard, "Isolation and characterization of a gene cluster involved in PAH degradation in Mycobacterium sp. strain SNP11: expression in Mycobacterium smegmatis mc(2)155," Research in microbiology, vol. 158, no. 2, pp. 175–186, 2007, doi: 10.1016/j.resmic.2006.11.002.
[6] K. Yuan et al., "Transcriptional response of Mycobacterium sp. strain A1-PYR to multiple polycyclic aromatic hydrocarbon contaminations," Environmental pollution (Barking, Essex : 1987), vol. 243, Pt B, pp. 824–832, 2018, doi: 10.1016/j.envpol.2018.09.001.
[7] S. Krivobok, S. Kuony, C. Meyer, M. Louwagie, J. C. Willison, and Y. Jouanneau, "Identification of pyrene-induced proteins in Mycobacterium sp. strain 6PY1: evidence for two ring-hydroxylating dioxygenases," Journal of bacteriology, vol. 185, no. 13, pp. 3828–3841, 2003, doi: 10.1128/jb.185.13.3828-3841.2003.
[8] M. M. Bradford, "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding," Analytical biochemistry, vol. 72, pp. 248–254, 1976, doi: 10.1006/abio.1976.9999.

@@ Line 6: / Line 6: @@
 __TOC__
+<br>
 ==Background==
-It is established that <html><href="https://parts.igem.org/Part:BBa_K5043009"><i>phtAc</i></a></html>,and <i>phtAd</i> as electron transfer components, along with a ring-hydroxylating dioxygenase system for example formed by <html><href="https://parts.igem.org/Part:BBa_K5043006"><i>pdoA2</i></a></html> and <html><href="https://parts.igem.org/Part:BBa_K5043007"><i>pdoB2</i></a></html>, aggregate to create a complex exhibiting dioxygenase activity. [4–6] This complex is capable of converting phenanthrene-4-carboxylate, an intermediate product of pyrene degradation, into <i>cis</i>-3,4-dihydroxy-phenanthrene-4-carboxylate, see Figure 1 [5, 6]. Given of this reaction’s importance in pyrene degradation, we opted to conduct a more detailed characterization. The component of the electron transport chain ferredoxin reductase <i>phtAd</i>, was produced and purified to elucidate their kinetic parameters. Subsequently, we aimed to analyze the complex formation with <i>pdoA2B2</i> through HPLC analysis.
+<html>
+  <p style="text-align: justify;">
+    It is well-established that <a href="https://parts.igem.org/Part:BBa_K5043009"><i>phtAc</i></a> and
+    <a href="https://parts.igem.org/Part:BBa_K5043010"><i>phtAd</i></a> together function as an electron transport chain, facilitating electron transfer from NADH [1].This system is crucial for supplying electrons to various dioxygenase systems, which are involved in the reduction of polycyclic aromatic hydrocarbons (PAHs). One such complex with dioxygenase activity is formed by <a href="https://https://parts.igem.org/Part:BBa_K5043001"><i>nidA</i></a> and <a href="https://https://https://parts.igem.org/Part:BBa_K5043002"><i>nidB</i></a>together with phtAc and phtAd, capable of degrading pyrene into <i>cis</i>-4,5-dihydroxy-4,5-dihydropyrene [2].
+ </p>
+</html>
+<html>
+  <p style="text-align: justify;">
+In this project, our complex of interest is formed by the ring-hydroxylating dioxygenase system, composed by <html><a href="https://parts.igem.org/Part:BBa_K5043006"><i>pdoA2</i> </a></html> and <html><a href="https://parts.igem.org/Part:BBa_K5043007"><i>pdoB2</i> </a></html>. Together with phtAcAd, these components aggregate to form a complex that exhibits dioxygenase activity [4–6]. This complex is capable of converting phenanthrene-4-carboxylate, an intermediate product of pyrene degradation, into <i>cis</i>-3,4-dihydroxy-phenanthrene-4-carboxylate, see Figure 1 [5, 6]. Given this reaction’s importance in pyrene degradation, we opted to conduct a more detailed characterization. The components of the electron transport chain, ferredoxin <i>phtAc</i> and ferredoxin reductase <i>phtAd</i>, were produced and purified to elucidate their kinetic parameters. Subsequently, we aimed to analyze the complex formation with <i>pdoA2B2</i> through HPLC analysis.
 <html><br>
          <figure>
            <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043010-fig1.png" style="width:100%">
-           <figcaption>Figure 1: Enzymatic reaction of <i>phtAcAd-pdoA2B2</i> complex. [3]<br>
+           <figcaption>Figure 1: Enzymatic reaction of <i>phtAcAd-pdoA2B2</i> complex [3].<br>
-<i>pdoA2B2 dimer exhibits catalytic side, which attacks substrate. phtAcAd transport two electrons from NADH to active side.</i></figcaption>
+<i><small>Proposed schema of a catalytic cycle mediated by pdoA2B2-PhtAcAd. Reaction: A molecule of dioxygen is integrated into polycyclic aromatic hydrocarbons (PAHs) through the transfer of two electrons supplied by NADH, facilitated by the electron transport complex phtAcAd.</small></i></figcaption>
          </figure></html>
 ==Enzyme production and purification==
+  <p style="text-align: justify;">
+Enzyme coding sequence was cloned into pQE bacterial expression vector with a N-terminal, hexahistidine tag. Proteins were expressed in <i>E. coli</i> BL21 (DE3). Main cultures were incubated at 37°C until an OD<small>600</small> of 0.5 was reached. Subsequently, cultures were induced with a final concentration of 0.5 mM IPTG and incubated overnight at 30°C. Purification was performed using Immobilized Metal Affinity Chromatography (IMAC). Both production and purification samples were analyzed via SDS-PAGE and Coomassie staining (data not shown). Enzyme concentration was determined using the Bradford Assay [8]. The final protein yield of <i>phtAd</i> was 105.1 µM.
-Enzyme coding sequences were cloned into pQE bacterial expression vector with a N-terminal, hexahistidine tag. Proteins were expressed in <i>E. coli</i> BL21 (DE3). Main cultures were incubated at 37°C until an OD600 of 0.5 was reached. Subsequently, cultures were induced with a final concentration of 0.5 mM IPTG and incubated overnight at 30°C. Purification was performed using Immobilized Metal Affinity Chromatography (IMAC). Both production and purification samples were analyzed via SDS-PAGE and coomassie staining (data not shown). Enzyme concentration was determined using the Bradford Assay. [7] <br>
+==Characterization of ferredoxin reductase <i>phtAd</i>==
+The characterization of <i>phtAd</i> activity was performed by evaluating its ability to reduce 2,6-dichlorophenolindophenol (DCPIP), which serves as an electron acceptor [1]. In its oxidized form, DCPIP displays a blue color, which transitions to colorless upon reduction, facilitating the evaluation of <i>phtAd</i> activity, see Figure 2. The decrease in absorbance was monitored at 600nm. NADH and FADH were needed as cofactors [1].
-==Characterization of ferredoxin reductase <i>phtAd</i> and ferredoxin <i>phtAc</i>==
+<html><br><br>
+        <figure>
-The characterization of <i>phtAd</i> (BBa_K5043010) activity was performed by evaluating its ability to reduce 2,6-dichlorophenolindophenol (DCPIP), which serves as an electron acceptor [3]. In its oxidized form, DCPIP displays a blue color, which transitions to colorless upon reduction, facilitating the evaluation of <i>phtAd</i> activity. The decrease in absorbance was monitored at 600nm. [3] <br>
+          <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043010-dcpip-1.png" style="width:70%">
-The activity of <i>phtAc</i> (BBa_K5043009) was assessed using a coupled assay. The increase in absorbance resulting from an electron transfer to cytochrome c signifies an interaction between ferredoxin <i>phtAc</i> and ferredoxin reductase <i>phtAd</i>. The increase in absorbance was monitored at 550nm. Both reactions needed NADH and FADH as cofactors . [3] <br>
+          <figcaption><br>Figure 2: Reduction of DCPIP performed with different <i>phtAd</i> concentrations.</br><br>
+</figcaption>
+        </figure><br></html>
 ===Method===
+The optimal reaction time and enzyme concentration for the assay were determined by measuring the decrease in absorbance at 2-minute intervals over a 30-minute period. This approach was necessary due to the uncertainty regarding the concentration at which the reaction would proceed and the duration for which the enzyme could effectively catalyze the reaction before reaching substrate saturation. Following the determination of these parameters, the reaction was assessed using varying concentrations of NADH while maintaining a constant enzyme concentration to determine the kinetic parameters. The reaction was initiated by the addition of a specified amount of <i>phtAd</i> and monitored at 600nm using a UV/Vis spectrometer.
-Initially, the optimal reaction time and enzyme concentration were established by measuring the decrease in absorbance over a 30-minute period at 2-minute intervals. Following the determination of these parameters, the reaction was assessed using varying concentrations of NADH while maintaining a constant enzyme concentration to determine the kinetic parameters. The reaction was initiated by the addition of a specified amount of <i>phtAd</i> and monitored at 600nm using a UV/Vis spectrometer.<br>
 ===Results===
+<b>Determination of the optimal enzyme concentration</b> <br>
+Initial optical analysis indicates that the assay conducted with the highest concentration of <i>phtAd</i>, 1.25 µM, resulted in the complete reduction of DCPIP, as evidenced by the solution's transition to a fully colorless state (see Figure 3). On the other hand, Figure 4 shows that there is no significant difference in the absorbance measurements between the assays conducted with 1 µM and 1.25 µM <i>phtAd</i>. However, the reaction conducted with 0.5 µM <i>phtAd</i> exhibited a lesser decrease in absorbance over the 30-minute period, indicating that the reaction proceeded at a relatively slow rate.<br>
+<html><br><br>
+  <style>
+    figure {
+      text-align: center; /* Center-aligns the figure */
+    }
-====Determination of the optimal enzyme concentration====
+    img {
+      width: 30%; /* Set image width */
+      border: 0.5px solid grey; /* Adds a frame to the image */
+    }
-Initial optical analysis indicates that the assay conducted with the highest concentration of <i>phtAd</i>, 1.25 µM, resulted in the complete reduction of DCPIP, as evidenced by the solution's transition to a fully colorless state (see Figure 2). On the other hand, Figure 3 shows that there is no significant difference in the absorbance measurements between the assays conducted with 1 µM and 1.25 µM <i>phtAd</i>. However, the reaction conducted with 0.5 µM <i>phtAd</i> exhibited a lesser decrease in absorbance over the 30-minute period, indicating that the reaction proceeded at a relatively slow rate.
+    figcaption {
+      margin-top: 0; /* Removes space between image and caption */
-<html><br><br>
+    }
-        <figure>
+  </style>
-          <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043010-fig2.png" style="width:100%">
-          <figcaption>Figure 2: Reduction of DCPIP with different <i>phtAd</i> concentration.</figcaption>
-        </figure><br></html>
-Regarding the optimal reaction time, Figure 3 illustrates that within the initial 10 minutes, absorbance decreases rapidly, indicating that the reduction of DCPIP occurs predominantly during this period. Beyond the 10-minute mark, absorbance measurements stabilize, suggesting that the reaction has reached a saturation point. This phenomenon may be attributed to the complete occupation of all enzyme’s active sites or the inability of the reaction to proceed further due to the lack of NADH regeneration.
+  <figure>
+    <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043010-fig2.png">
+    <figcaption>Figure 3: Reduction of DCPIP with different <i>phtAd</i> concentrations [μM].</figcaption>
+  </figure>
+<br></html>
+Regarding the optimal reaction time, Figure 4 illustrates that within the initial 10 minutes, absorbance decreases rapidly, indicating that the reduction of DCPIP occurs predominantly during this period. Beyond the 10-minute mark, absorbance measurements stabilize, suggesting that the reaction has reached a saturation point. This phenomenon may be attributed to the complete reduction of DCPIP or the inability of the reaction to proceed further due to the lack of NADH regeneration.<br>
 <html><br><br>
          <figure>
-           <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043010-fig3.png" style="width:100%">
+           <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043010-fig3.png" style="width:70%">
-           <figcaption>Figure 3: Reduction of DCPIP performed with Different <i>phtAd</i> concentrations.<br>
+           <figcaption>Figure 4: Reduction of DCPIP performed with different <i>phtAd</i> concentrations.<br>
 <i>The reduction of DCPIP by <i>phtAd</i> was monitored for 30 min at a constant substrate quantity and varying amount of enzyme. The reaction mixture contained 100 mM Tris/HCl (pH 7.5), 100 µM NADH, 30 µM DCPIP, 0.5 µM FAD and 0.5, 1, 1.25 µM <i>phtAd</i>. Reaction was performed at room temperature.</i></figcaption>
          </figure><br></html>
-Based on the results obtained, we selected a concentration of 1.25 µM <i>phtAd</i> for the subsequent assay, as this concentration demonstrated a complete reduction of DCPIP.<br>
-====Kinetic parameters of <i>phtAd</i>====
-For the determination of the kinetic parameters the assay was run with different NADH concentrations ranging from 40 µM to 250 µM. Data obtained from these assays were then analyzed using Michaelis-Menten kinetics (see Figure 4) and a Lineweaver-Burk plot (see Figure 5) was constructed to provide a linear representation of the enzyme kinetics to determine key parameters such as Vmax and Km.
+Based on the results obtained, we selected a concentration of 1.25 µM <i>phtAd</i> for the subsequent assay, as this concentration demonstrated a complete reduction of DCPIP. <br>
+===Kinetic parameters of <i>phtAd</i>===
+For the determination of kinetic parameters the assay was run with different NADH concentrations ranging from 40 µM to 250 µM. Data obtained from these assays was then analyzed using Michaelis-Menten kinetics (see Figure 5) and a Lineweaver-Burk plot (see Figure 6) was constructed to provide a linear representation of enzyme kinetics to determine key parameters such as Vmax and Km. <br>
 <html><br><br>
          <figure>
-           <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043010-fig4.png" style="width:100%">
+           <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043010-fig4.png" style="width:60%">
-           <figcaption>Figure 4: Michaelis-Menten Curve for <i>phtAd</i>.<br>
+           <figcaption>Figure 5: Michaelis-Menten Curve for <i>phtAd</i>.<br>
-  <i>The velocity was calculated utilizing the Lambert-Beer Law with the extinction coefficient of cytochrome c (ε600 23 mM-1 x cm–1</i>) [3]<i>. A logarithmic trend line was applied as a preliminary approximation.</i></figcaption>
+  <i>The velocity <i><small>v = ΔE / (ε × d × Δt)</small></i> was calculated utilizing the Lambert-Beer Law with the extinction coefficient of DCPIP (ε600 23 mM-1 x cm–1</i>) [1]<i>. A logarithmic trend line was applied as a preliminary approximation.</i><br></figcaption>
          </figure><br></html>
 <html><br><br>
          <figure>
-           <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043010-fig5.png" style="width:100%">
+           <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043010-fig5.png" style="width:60%">
-           <figcaption>Figure 5: Lineweaver-Burk plot.<br>
+           <figcaption>Figure 6: Lineweaver-Burk plot.<br>
   <i>Graphs show the fitted linear function and its correlation coefficient.</i></figcaption>
          </figure><br></html>
+The <b>Vmax, Km, kcat, kcat/Km values</b> of ferredoxin reductase <i>phtAd</i> were <b>0.0011 mM/min, 0.028 mM, 1.12 min<sup>-1</sup> and 39.46 mM<sup>-1</sup> x min<sup>-1</sup></b>, respectively.  Notable discrepancies from the values reported in the literature [1] were identified, which can be attributed to variations in enzyme concentration. The reaction was performed using a higher enzyme concentration due to the observed diminished activity of the produced enzymes.<br>
-The Vmax, Km, kcat, kcat/Km values of the ferredoxin reductase <i>phtAd</i> were 0.0011 mM/min, 0.028 mM, 1.12 min-1 and 39.46 mM-1 x min-1, respectively.  Notable discrepancies from the values reported in the literature [3] were identified, which can be attributed to variations in enzyme concentration. The reaction was performed using a higher enzyme concentration due to the observed diminished activity of the produced enzymes.
+Furthermore, the R<sup>2</sup> value of 0.4668 indicates that the Lineweaver-Burk plot does not perfectly describe the relationship between 1/[NADH] and 1/V. While the fit is moderate, it suggests that factors like experimental variability, biological complexity, or non-linearity in enzyme behavior might be contributing to deviations from the ideal linear relationship.
-==Kinetic characterization of <i>phtAc</i> coupled to <i>phtAd</i>==
+==Characterization of ferredoxin <i>phtAc</i> coupled to <i>phtAd</i>==
-===Method===
-Initial measurements were conducted in accordance with the methodology outlined by Wu et al. (2020) [1]. However, the assay produced inconclusive results, as no variation in absorption was observed over time. This lack of change was attributed to the high concentration of cytochrome c (600 µM) utilized in the initial trials. Consequently, we opted to conduct further assays employing varying concentrations of cytochrome c and NADH to identify the optimal concentrations that would facilitate a measurable increase in absorption. In alignment with the approach taken by Wu et al. (2020) [1], subsequent assays were performed using a <i>phtAd</i> to <i>phtAc</i> ratio of 1:3. The increase in absorbance was measured over a 30-minute period at 2-minute intervals at 550 nm. After the optimal cytochrome c concentration was established, further assays were performed varying the NADH concentration to determine the kinetic parameters. <br>
+The activity of <i>phtAc</i> was assessed using a coupled assay. The increase in absorbance resulting from an electron transfer to cytochrome c (electron acceptor) signifies an interaction between ferredoxin <i>phtAc</i> and ferredoxin reductase <i>phtAd</i>. The increase in absorbance was monitored at 550nm. NADH and FADH are needed as cofactors. [1] <br>
+===Method===
+Initial measurements were conducted in accordance with the methodology outlined by Wu et al. (2020) [1]. However, the assay produced inconclusive results, as no variation in absorption was observed over time. This lack of change was attributed to the high concentration of cytochrome c (600 µM) utilized in the initial trials. Consequently, we opted to conduct further assays employing varying concentrations of cytochrome c and NADH to identify the optimal concentrations that would facilitate a measurable increase in absorption. In alignment with the approach taken by Wu et al. (2020) [1], subsequent assays were performed using a <i>phtAd</i> to <i>phtAc</i> ratio of 1:3. The increase in absorbance was measured over a 30-minute period at 2-minute intervals at 550 nm. After the optimal cytochrome c concentration was established, further assays were performed varying the NADH concentration to determine the kinetic parameters. <br>
 ===Results===
 <b>Determination of the optimal substrate concentration</b><br>
-In the initial assay conducted with 250 µM cytochrome c, no change in absorbance was observed (see Figure 6A). Considering this outcome, the subsequent assay was performed using 100 µM cytochrome c in conjunction with a higher concentration of NADH (200 µM). This combination resulted in a measurable increase in absorbance, demonstrating a linear progression over a duration of 26 minutes (see Figure 6C) indicating the oxidation of cytochrome c. To validate this outcome, additional assays were conducted utilizing 100 µM cytochrome c alongside varying concentrations of NADH, both higher and lower. The assay employing 250 µM NADH also demonstrated a linear increase in absorption (see Figure 6B) in the first 26 minutes. Conversely, the assay with 100 µM NADH (See Figure 6D) also exhibited a linear increase in absorption, albeit at a reduced rate. Notably, no saturation point was observed at this concentration, in contrast to the higher concentrations, which indicated saturation occurring approximately 30 minutes into the assay.<br>
+In the initial assay conducted with 250 µM cytochrome c, no change in absorbance was observed (see Figure 7A). Considering this outcome, the subsequent assay was performed using 100 µM cytochrome c in conjunction with a higher concentration of NADH (200 µM). This combination resulted in a measurable increase in absorbance, demonstrating a linear progression over a duration of 26 minutes (see Figure 7C) indicating the oxidation of cytochrome c. To validate this outcome, additional assays were conducted utilizing 100 µM cytochrome c alongside varying concentrations of NADH, both higher and lower. The assay employing 250 µM NADH also demonstrated a linear increase in absorption (Figure 7B) in the first 26 minutes. Conversely, the assay with 100 µM NADH (See Figure 7D) also exhibited a linear increase in absorption, albeit at a reduced rate. Notably, no saturation point was observed at this concentration, in contrast to the higher concentrations, which indicated saturation occurring approximately 30 minutes into the assay.<br>
 <html><br><br>
          <figure>
            <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043009-fig1.png" style="width:90%">
-           <figcaption>Figure 6: Oxidation of Cytochrome c catalyzed by <i>phtAc</i>, in conjunction with <i>phtAd</i>, under varying concentrations of Cytochrome c and NADH.<br>
+           <figcaption>Figure 7: Oxidation of cytochrome c catalyzed by <i>phtAc</i>, in conjunction with <i>phtAd</i>, under varying concentrations of cytochrome c and NADH.<br>
 <i>The oxidation of cytochrome c by phtAc coupled with phtAd was monitored for 30 min at a constant enzyme concentration and varying amount of substrate. The reaction mixture contained 100 mM Tris/HCl (pH 7.5), 100 – 250 µM NADH, 100 and 250 µM cyt. c, 0.5 µM FAD, 1 µM phtAd and 3.5 µM phtAc. Reaction was performed at room temperature.</i></figcaption>
          </figure><br></html>
+<br>
 Based on the results obtained, we chose a concentration of 100 µM cytochrome c for the subsequent tests, as this concentration showed a measurable increase in absorbance. For the reaction time, we decided to run further reactions for 34 minutes to confirm the saturation point. <br>
 ===Kinetic parameters of <i>phtAc</i>===
+For the determination of kinetic parameters the assay was run with different NADH concentrations ranging from 40 µM to 400 µM. Data obtained from these assays was then analyzed using Michaelis-Menten kinetics (see Figure 8) and a Lineweaver-Burk plot (see Figure 9) was constructed to provide a linear representation of the enzyme kinetics to determine key parameters such as Vmax and Km. <br>
-For the determination of the kinetic parameters the assay was run with different NADH concentrations ranging from 40 µM to 400 µM. Data obtained from these assays were then analyzed using Michaelis-Menten kinetics (see Figure 7) and a Lineweaver-Burk plot (see Figure 8) was constructed to provide a linear representation of the enzyme kinetics to determine key parameters such as Vmax and Km. <br>
- <br>
 <html><br><br>
          <figure>
-           <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043009-fig2.png" style="width:70%">
+           <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043009-fig2.png" style="width:60%">
-           <figcaption>Figure 7: Michaelis-Menten Curve for PhtAc.
+           <figcaption>Figure 8: Michaelis-Menten curve for <i>phtAc.<br>
-<i>The velocity was calculated utilizing the Lambert-Beer Law with the extinction coefficient of Cytochrome C (ε550 21 mM-1 x cm–1) [6]. A logarithmic trend line was applied as a preliminary approximation.</i></figcaption>
+The velocity <i><small>v = ΔE / (ε × d × Δt)</small></i>was calculated utilizing the Lambert-Beer Law with the extinction coefficient of cytochrome C (ε550 21 mM<sup>-1</sup> x cm<sup>–1</sup>) [6]. A logarithmic trend line was applied as a preliminary approximation.</i></figcaption>
          </figure><br></html>
-<html><br><br>
+<html>
          <figure>
-           <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043009-fig3.png" style="width:70%">
+           <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043009-fig3.png" style="width:60%">
-           <figcaption>Figure 8: Lineweaver-Burk plot.
+           <figcaption>Figure 9: Lineweaver-Burk plot.<br>
 <i>Graphs show the fitted linear function and its correlation coefficient.</i></figcaption>
          </figure><br></html>
+The <b>Vmax, Km, kcat, kcat/Km values</b> of the ferredoxin <i>phtAc</i> were <b>0.00043 mM/min, 0.31 mM, 0.12 1/min and 3.9 1/mM x min</b>, respectively.  Significant deviations from the literature values [3] were observed; however, the reaction was conducted with a substantially lower concentration of cytochrome c and elevated concentrations of enzymes, as the produced enzymes exhibited reduced activity.<br>
+Moreover, the R<sup>2</sup> value of 0.5378 derived from the kinetic analysis of <i>phtAc</i> is marginally higher than that of <i>phtAd</i>. This marginally elevated value may indicate a greater reliability of the data or a more consistent enzymatic response across varying NADH concentrations. Nevertheless, the Lineweaver-Burk plot does not accurately characterize the relationship between 1/[NADH] and 1/V. Although the fit is moderate, it implies that factors such as experimental variability, biological complexity, or non-linear enzyme behavior could be responsible for the observed deviations from the expected linear relationship.
+<br>
 ==Activity assay coupled with HPLC analysis==
-We successfully characterized and demonstrated the activity of <i>phtAc</i> and <i>phtAd</i> both individually and as a complex. This complex functions as an electron carrier for <i>pdoA2B2</i>  [4–6]. The subsequent step involved conducting an activity assay in conjunction with HPLC analysis with the aim to evaluate the function of <i>pdoA2B2</i> both independently and in complex with <i>phtAcAd</i>, forming a tetramer. <br>
+We successfully characterized and demonstrated the activity of <i>phtAc</i> and <i>phtAd</i> both individually and as a complex. This complex functions as an electron carrier for <i>pdoA2B2</i> [4–6]. The subsequent step involved conducting an activity assay in conjunction with HPLC analysis with the aim to evaluate the function of <i>pdoA2B2</i> both independently and in complex with <i>phtAcAd</i>, forming a tetramer. <br>
 ===Method===
-Initially, the activity assay was conducted, wherein the enzymes were incubated at 30°C for approximately 15 to 20 minutes, both in the presence and absence of NADH to examine the effect of NADH on enzymatic aggregation. Subsequently, phenanthrene-4-carboxylate (P4C) was added to initiate the reaction. The reaction mixture was then incubated for 30 minutes at 30°C, after which the reaction was terminated by the addition of 100% methanol. Following this, HPLC analysis was performed using a Zorbax SB-C18 column, employing an acetonitrile/water gradient at a flow rate of 0.4 ml/min, with the column oven maintained at room temperature. The gradient elution program commenced with an initial mobile phase of 60:40 (v/v) acetonitrile to water, transitioning linearly to 100% acetonitrile over 14 minutes, followed by a return to the initial phase (60:40) after 5 minutes. The total duration for each analysis was 35 minutes. For the negative control, an assay was conducted using denatured enzymes, achieved by heating the enzymes at 95°C for 10 minutes. <br>
+Initially, the activity assay was conducted, wherein the enzymes were incubated at 30°C for approximately 15 to 20 minutes, both in the presence and absence of NADH to examine the effect of NADH on enzymatic aggregation. Subsequently, phenanthrene-4-carboxylate (P4C) was added to initiate the reaction. The reaction mixture was then incubated for 30 minutes at 30°C, after which the reaction was terminated by the addition of 100% methanol. Following this, HPLC analysis was performed using a Zorbax SB-C18 column, employing an acetonitrile/water gradient at a flow rate of 0.4 ml/min, with the column oven maintained at room temperature. The gradient elution program commenced with an initial mobile phase of 60:40 (v/v) acetonitrile to water, transitioning linearly to 100% acetonitrile over 14 minutes, followed by a return to the initial phase (60:40) after 5 minutes. The total duration for each analysis was 35 minutes. For the negative control, an assay was conducted using denatured enzymes, achieved by heating the enzymes at 95°C for 10 minutes. Peaks were analyzed at 270 nm absorbance.<br>
 ===Results===
 Initial activity assays were performed solely with <i>pdoA2</i> and <i>pdoB2</i> to form a dimer, thereby validating their enzymatic activity and evaluating their aggregation characteristics. Monomers were incubated together for aggregation in the presence and absence of NADH to determine whether NADH, as a cofactor, influences the aggregation process. <br>
-The efficacy of the reaction was assessed by measuring the absorbance of phenanthrene-4-carboxylate (P4C) and NADH using HPLC. The area under the resulting peaks corresponding to the levels of P4C and NADH was determined, enabling a comparative analysis of the consumption of P4C and NADH against established standards and between the samples. Furthermore, to eliminate potential statistical errors in the assessment of our results, we employed triplicate measurements for each sample and calculated their averages. The results are presented in Figure 9. Results indicated that the assays in which NADH was added prior to aggregation exhibited enhanced P4C degradation and, correspondingly, increased NADH consumption compared to the assays where NADH was added after aggregation. However, the observed NADH consumption was relatively low, which may be attributed to the absence of the electron carrier dimer (<i>phtAcAd</i>) in the sample, thereby limiting NADH consumption.
+The efficacy of the reaction was assessed by measuring the absorbance of phenanthrene-4-carboxylate (P4C) and NADH using HPLC. The area under the resulting peaks corresponding to the levels of P4C and NADH was determined, enabling a comparative analysis of the consumption of P4C and NADH against established standards and between the samples. In this context, phenanthrene-4-carboxylate consumption refers to the extent of P4C degradation, as indicated by a reduction in peak area compared to the standard. In the absence of additional samples with different concentrations, it is not possible to quantify the remaining P4C in the samples. The only conclusion that can be drawn is whether P4C has been degraded, as evidenced by the decrease in the area of the corresponding peak; this applies also to NADH.
+Furthermore, to eliminate potential statistical errors in the assessment of our results, we employed triplicate measurements for each sample and calculated their averages. The results are presented in Figure 10. Results indicated that the assays in which NADH was added prior to aggregation exhibited enhanced P4C degradation and, correspondingly, increased NADH consumption compared to the assays where NADH was added after aggregation. However, the observed NADH consumption was relatively low, which may be attributed to the absence of the electron carrier dimer (<i>phtAcAd</i>) in the sample, thereby limiting NADH consumption. <br>
 <html><br><br>
          <figure>
-           <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043010-fig6.png" style="width:100%">
+           <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043010-fig10.png" style="width:100%">
-           <figcaption>Figure 9: Comparative Analysis of phenanthrene-4-carboxylate (P4C) and NADH Consumption in <i>pdoA2</i>:<i>pdoB2</i> activity assays.<br>
+           <figcaption>Figure 10: Comparative Analysis of phenanthrene-4-carboxylate (P4C) and NADH Consumption in <i>pdoA2</i>:<i>pdoB2</i> activity assays.<br>
-<i>The degradation of P4C by <i>pdoA2</i>:<i>pdoB2</i> was monitored for 30 min at a constant substrate and enzyme concentration.  The reaction mixture contained 100 mM Tris/HCl (pH 7.5), 25 µM P4C, 1 µM <i>pdoA2</i> and 1 µM <i>pdoB2</i>. The 100 µM NADH were either added after aggregation (a.A) or before aggregation (b.A). </i></figcaption>
+<i>The degradation of P4C by <i>pdoA2</i>:<i>pdoB2</i> was monitored for 30 min at a constant substrate and enzyme concentration. The reaction mixture contained 100 mM Tris/HCl (pH 7.5), 25 µM P4C, 1 µM <i>pdoA2</i> and 1 µM <i>pdoB2</i>. The 100 µM NADH were either added after or before aggregation. </i></figcaption>
          </figure><br></html>
-Considering the initial results, the analysis of the tetramer aggregation process and the degradation of phenanthrene-4-carboxylat was conducted by introducing NADH prior to the aggregation process. <br><br>
+Considering the initial results, the analysis of the tetramer aggregation process and the degradation of phenanthrene-4-carboxylate was conducted by introducing NADH prior to the aggregation process. <br><br>
-<b>Degradation of phenanthrene-4-carboxylat by enzyme complex with dioxygenase activity using NADH as a cofactor</b><br>
+<b>Degradation of phenanthrene-4-carboxylate by enzyme complex with dioxygenase activity using NADH as a cofactor</b>  <br>
-The presence of the functional enzyme complex with dioxygenase activity is anticipated to result in a reduction in the quantity of phenanthrene-4-carboxylate. This reduction can be quantified by determining the decrease in the area of the corresponding peak in comparison to the standard. Given the unknown stoichiometry of this enzyme complex, we conducted assays utilizing two distinct stoichiometric ratios. The first assay employed a 1:1 stoichiometry of all monomers, while the second utilized a ratio of <i>pdoA2<sup>1</sup></i>:<i>pdoB2<sup>1</sup></i>:<i>phtAc</i><sup>3</sup>:<i>phtAd<sup>1</sup></i>. This latter stoichiometry was selected based on previous kinetic analyses of <i>phtAc</i>, which were performed with a ratio of three <i>phtAc</i> molecules to one <i>phtAd</i> for determining kinetic parameters. This ratio was employed in the present assays to assess its influence on the degradation of phenanthrene-4-carboxylate and to determine whether the presence of three <i>phtAc</i> molecules enhanced the efficiency of the electron transfer process, thereby accelerating the breakdown of phenanthrene-4-carboxylate. However, results showed (see
-Figure 10) that the breakdown of phenanthrene-4-carboxylate was slightly more efficient with the 1:1 ratio of the monomers; this could suggest that a higher ratio of <i>phtAc</i> does not enhance the electron transfer process. And that the 1:1 ratio may have created a more favorable environment for the interaction of the active site with the substrate. <br>
-To confirm the aggregation of the enzyme complex and the successful degradation of P4C by this complex, negative controls were prepared. For this, assays were prepared with denatured enzymes with the stoichiometries described above. Results showed (see Figure 10) that the peak area of P4C within these assays was higher in comparison with the assays with the active enzyme complex.  The comparison of these results indicates that the enzyme complex was successfully formed and demonstrated the capacity to degrade P4C.
+The presence of the functional enzyme complex with dioxygenase activity is anticipated to result in a reduction in the quantity of phenanthrene-4-carboxylate. This reduction can be quantified by determining the decrease in the area of the corresponding peak in comparison to the standard. Given the unknown stoichiometry of this enzyme complex, we conducted assays utilizing two distinct stoichiometric ratios. The first assay employed a 1:1 stoichiometry of all monomers, while the second utilized a ratio of <i>pdoA2<sup>1</sup></i>:<i>pdoB2<sup>1</sup></i>:<i>phtAc</i><sup>3</sup>:<i>phtAd<sup>1</sup></i>. This latter stoichiometry was selected based on previous kinetic analyses of <i>phtAc</i>, which were performed with a ratio of three equivalents <i>phtAc</i> to one <i>phtAd</i> for determining kinetic parameters. [3] This ratio was employed in the present assays to assess its influence on the degradation of phenanthrene-4-carboxylate and to determine whether the presence of three equivalents <i>phtAc</i> enhanced the efficiency of the electron transfer process, thereby accelerating the breakdown of phenanthrene-4-carboxylate. However, results showed (see Figure 11) that the breakdown of phenanthrene-4-carboxylat was slightly more efficient with the 1:1 ratio of the monomers; this could suggest that a higher ratio of <i>phtAc</i> does not enhance the electron transfer process. And that the complex with 1:1 ratio of all enzymes aggregates better. <br>
+To confirm the aggregation of the enzyme complex and the successful degradation of P4C by this complex, negative controls were prepared. For this, assays were prepared with denatured enzymes with the stoichiometries described above. Results showed (see Figure 11) that the peak area of P4C within these assays was higher in comparison with the assays with the active enzyme complex.  The comparison of these results indicates that the enzyme complex was successfully formed and demonstrated the capacity to degrade P4C. <br>
 <html><br><br>
          <figure>
-           <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043010-fig7.png" style="width:100%">
+           <img src="https://static.igem.wiki/teams/5043/registry/bba-k5043010-fig11-1.png" style="width:100%">
-           <figcaption>Figure 10: Comparative Analysis of phenanthrene-4-carboxylat (P4C) and NADH Consumption in <i>pdoA2</i>:<i>pdoB2</i>:<i>phtAc</i>:<i>phtAd</i> activity assays.<br>
+           <figcaption>Figure 11: Comparative Analysis of phenanthrene-4-carboxylate (P4C) and NADH Consumption in <i>pdoA2</i>:<i>pdoB2</i>:<i>phtAc</i>:<i>phtAd</i> activity assays.<br>
 <i>The degradation of P4C by the enzyme complex was monitored for 30 min at a constant substrate and enzyme concentration. The reaction mixture contained 100 mM Tris/HCl (pH 7.5), 200 µM NADH, 40 µM P4C, 1 µM pdoA2, 1 µM pdoB2, 1 or 3 µM and 1 µM phtAd</i>.</figcaption>
          </figure><br></html>
-Another negative control was conducted by excluding any enzymes from the assay.  In comparison to samples containing denatured enzymes, the results showed a reduced peak area in the peak corresponding to phenanthrene-4-carboxylate, suggesting either a diminished quantity of P4C in the sample or its degradation. However, the possibility of cross-reactivity with other components in the assay can be discounted, as such interactions would also manifest in the other negative controls. This observation indicates the presence of an independent source of error. Potential issues may involve the unintentional introduction of enzymes or inadequate substrate concentration. The presence of enzymes in the assay can be corroborated by taking into consideration the amount of NADH; notably, the peak area of NADH in this sample was diminished suggesting the possibility that a reaction has taken place, and the results are more consistent with those obtained from the assay conducted with a 1:3 enzyme ratio (see
-Figure 10). While numerous errors could have arisen, a systematic issue such as cross-reactivity, which would render the enzyme ineffective, can be dismissed by comparing the sample to those with non-functional enzymes. <br>
-Moreover, as anticipated, the samples containing the active enzyme complex demonstrated a decrease in the peak area associated with NADH relative to the standard (see
+Another negative control was conducted by excluding any enzymes from the assay.  In comparison to samples containing denatured enzymes, the results showed a reduced peak area in the peak corresponding to phenanthrene-4-carboxylate, suggesting either a diminished quantity of P4C in the sample or its degradation. However, the possibility of cross-reactivity with other components in the assay can be discounted, as such interactions would also manifest in the other negative controls. This observation indicates the presence of an independent source of error. Potential issues may involve the unintentional introduction of enzymes or inadequate substrate concentration. The presence of enzymes in the assay can be corroborated by taking into consideration the amount of NADH; notably, the peak area of NADH in this sample was diminished suggesting the possibility that a reaction has taken place, and the results are more consistent with those obtained from the assay conducted with a 1:3 enzyme ratio (see Figure 11). While numerous errors could have arisen, a systematic issue such as cross-reactivity, which would render the enzyme ineffective, can be dismissed by comparing the sample to those with non-functional enzymes. <br>
-Figure 10). This finding corroborates the functionality of the electron carrier dimer, which collaborates with <i>pdoA2B2</i> in the degradation of P4C. In contrast, the assay involving denatured enzymes at a 1:1 ratio revealed no consumption of NADH, indicating the absence of any reaction. <br>
-However, if we observed the results of NADH consumption from the assay involving denatured enzymes, utilizing three molecules of <i>phtAc</i> in conjunction with a 1:1 ratio of the other enzymes, demonstrate a reduction in NADH consumption, as evidenced by the diminished area of the corresponding peak relative to the standard. This observation indicates the presence of an independent source of error, as the other assay performed with denatured enzymes shows no NADH consumption. Potential issues may include insufficient NADH concentration. If the NADH concentration was low, degradation could have occurred over time, as the activity assays were conducted days prior to the HPLC analysis. The thawing process and exposure of the sample to temperature fluctuations may have contributed to NADH degradation due to its inherent instability. Additionally, the possibility of incomplete denaturation should be considered. If the enzymes were not fully denatured during the assay, residual active enzyme could remain, resulting in NADH consumption. The denaturation conditions may not have been adequate to completely inactivate the enzymes. This is further supported by the observation that P4C consumption was slightly lower in comparison to the sample with denatured enzymes with the 1:1 ratio. <br>
+Moreover, as anticipated, the samples containing the active enzyme complex demonstrated a decrease in the peak area associated with NADH relative to the standard (see Figure 11). This finding corroborates the functionality of the electron carrier dimer, which collaborates with <i>pdoA2B2</i> in the degradation of P4C. In contrast, the assay involving denatured enzymes at a 1:1 ratio revealed no consumption of NADH, indicating the absence of any reaction. <br>
+However, the results of NADH consumption from the assay involving denatured enzymes, utilizing three equivalents of <i>phtAc</i> in conjunction with a 1:1 ratio of the other enzymes, demonstrate a reduction in NADH consumption, as evidenced by the diminished area of the corresponding peak relative to the standard. This observation indicates the presence of an independent source of error, as the other assay performed with denatured enzymes shows no NADH consumption. Potential issues may include insufficient NADH concentration. If the NADH concentration was low, degradation could have occurred over time, as the activity assays were conducted days prior to the HPLC analysis. The thawing process and exposure of the sample to temperature fluctuations may have contributed to NADH degradation due to its inherent instability. Additionally, the possibility of incomplete denaturation should be considered. If the enzymes were not fully denatured during the assay, residual active enzyme could remain, resulting in NADH consumption. The denaturation conditions may not have been adequate to completely inactivate the enzymes. This is further supported by the observation that P4C consumption was slightly lower in comparison to the sample with denatured enzymes with the 1:1 ratio.
 ==Conclusion==
-In conclusion, the enzyme complex with dioxygenase activity demonstrated its ability to degrade phenanthrene-4-carboxylate (P4C), with slightly greater efficiency observed in the assay utilizing a 1:1 ratio of monomers compared to the 1:3 ratio of <i>phtAc</i> to <i>pdoA2</i>:<i>pdoB2</i>:<i>phtAd</i>. This suggests that a higher ratio of <i>phtAc</i> does not enhance electron transfer efficiency. Negative controls, including assays with denatured enzymes and no enzymes, confirmed that the active enzyme complex was responsible for P4C degradation, while the reduction in NADH in some denatured enzyme assays points to possible errors, such as NADH degradation or incomplete enzyme denaturation. Further assays with stricter controls are needed to confirm these findings and minimize potential sources of error. <br>
+In conclusion, the enzyme complex with dioxygenase activity demonstrated its ability to degrade phenanthrene-4-carboxylate (P4C), with slightly greater efficiency observed in the assay utilizing a 1:1 ratio of monomers compared to the 1:3 ratio of <i>phtAc</i> to <i>pdoA2</i>:<i>pdoB2</i>:<i>phtAd</i>. This suggests that a higher ratio of <i>phtAc</i> does not enhance electron transfer efficiency. Negative controls, including assays with denatured enzymes and no enzymes, confirmed that the active enzyme complex was responsible for P4C degradation, while the reduction in NADH in some denatured enzyme assays points to possible errors, such as NADH degradation or incomplete enzyme denaturation. Further assays with stricter controls are needed to confirm these findings and minimize potential sources of error.
@@ Line 175: / Line 204: @@
 == References ==
 <small>
-[1]	L. Guo et al., "Characterization of a novel aromatic ring-hydroxylating oxygenase, NarA2B2, from thermophilic Hydrogenibacillus sp. strain N12," Applied and environmental microbiology, vol. 89, no. 10, e0086523, 2023, doi: 10.1128/aem.00865-23.
+[1]	Y. Wu, Y. Xu, and N. Zhou, "A newly defined dioxygenase system from Mycobacterium vanbaalenii PYR-1 endowed with an enhanced activity of dihydroxylation of high-molecular-weight polyaromatic hydrocarbons," Front. Environ. Sci. Eng., vol. 14, no. 1, 2020, doi: 10.1007/s11783-019-1193-5.
-[2]	S.-J. Kim, O. Kweon, R. C. Jones, J. P. Freeman, R. D. Edmondson, and C. E. Cerniglia, "Complete and integrated pyrene degradation pathway in Mycobacterium vanbaalenii PYR-1 based on systems biology," Journal of bacteriology, vol. 189, no. 2, pp. 464–472, 2007, doi: 10.1128/JB.01310-06.
-[3]	Y. Wu, Y. Xu, and N. Zhou, "A newly defined dioxygenase system from Mycobacterium vanbaalenii PYR-1 endowed with an enhanced activity of dihydroxylation of high-molecular-weight polyaromatic hydrocarbons," Front. Environ. Sci. Eng., vol. 14, no. 1, 2020, doi: 10.1007/s11783-019-1193-5.
+[2]	Kweon, O., Kim, S., Freeman, J. P., Song, J., Baek, S., & Cerniglia, C. E, "Substrate Specificity and Structural Characteristics of the Novel Rieske Nonheme Iron Aromatic Ring-Hydroxylating Oxygenases NidAB and NidA3B3 from Mycobacterium vanbaalenii PYR-1" mBio, 1(2), 2010, doi: 10.1128/mbio.00135-10
-[4]	C. Pagnout, G. Frache, P. Poupin, B. Maunit, J.-F. Muller, and J.-F. Férard, "Isolation and characterization of a gene cluster involved in PAH degradation in Mycobacterium sp. strain SNP11: expression in Mycobacterium smegmatis mc(2)155," Research in microbiology, vol. 158, no. 2, pp. 175–186, 2007, doi: 10.1016/j.resmic.2006.11.002.
-[5]	K. Yuan et al., "Transcriptional response of Mycobacterium sp. strain A1-PYR to multiple polycyclic aromatic hydrocarbon contaminations," Environmental pollution (Barking, Essex : 1987), vol. 243, Pt B, pp. 824–832, 2018, doi: 10.1016/j.envpol.2018.09.001.
+[3]     S.-J. Kim, O. Kweon, R. C. Jones, J. P. Freeman, R. D. Edmondson, and C. E. Cerniglia, "Complete and integrated pyrene degradation pathway in Mycobacterium vanbaalenii PYR-1 based on systems biology," Journal of bacteriology, vol. 189, no. 2, pp. 464–472, 2007, doi: 10.1128/JB.01310-06.<br>
-[6]	S. Krivobok, S. Kuony, C. Meyer, M. Louwagie, J. C. Willison, and Y. Jouanneau, "Identification of pyrene-induced proteins in Mycobacterium sp. strain 6PY1: evidence for two ring-hydroxylating dioxygenases," Journal of bacteriology, vol. 185, no. 13, pp. 3828–3841, 2003, doi: 10.1128/jb.185.13.3828-3841.2003.
+[4]	L. Guo et al., "Characterization of a novel aromatic ring-hydroxylating oxygenase, NarA2B2, from thermophilic Hydrogenibacillus sp. strain N12," Applied and environmental microbiology, vol. 89, no. 10, e0086523, 2023, doi: 10.1128/aem.00865-23.<br>
-[7]	M. M. Bradford, "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding," Analytical biochemistry, vol. 72, pp. 248–254, 1976, doi: 10.1006/abio.1976.9999.
+[5]	C. Pagnout, G. Frache, P. Poupin, B. Maunit, J.-F. Muller, and J.-F. Férard, "Isolation and characterization of a gene cluster involved in PAH degradation in Mycobacterium sp. strain SNP11: expression in Mycobacterium smegmatis mc(2)155," Research in microbiology, vol. 158, no. 2, pp. 175–186, 2007, doi: 10.1016/j.resmic.2006.11.002.<br>
+[6]	K. Yuan et al., "Transcriptional response of Mycobacterium sp. strain A1-PYR to multiple polycyclic aromatic hydrocarbon contaminations," Environmental pollution (Barking, Essex : 1987), vol. 243, Pt B, pp. 824–832, 2018, doi: 10.1016/j.envpol.2018.09.001.<br>
+[7]	S. Krivobok, S. Kuony, C. Meyer, M. Louwagie, J. C. Willison, and Y. Jouanneau, "Identification of pyrene-induced proteins in Mycobacterium sp. strain 6PY1: evidence for two ring-hydroxylating dioxygenases," Journal of bacteriology, vol. 185, no. 13, pp. 3828–3841, 2003, doi: 10.1128/jb.185.13.3828-3841.2003.<br>
+[8]	M. M. Bradford, "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding," Analytical biochemistry, vol. 72, pp. 248–254, 1976, doi: 10.1006/abio.1976.9999.
 </small>

Difference between revisions of "Part:BBa K5043010"

Latest revision as of 09:22, 2 October 2024

Contents

Background

Enzyme production and purification

Characterization of ferredoxin reductase phtAd

Method

Results

Kinetic parameters of phtAd

Characterization of ferredoxin phtAc coupled to phtAd

Method

Results

Kinetic parameters of phtAc

Activity assay coupled with HPLC analysis

Method

Results

Conclusion

References