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.

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].

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.

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.

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.

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.

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.

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.

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.

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

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.

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