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phtAc from M. vanbaalenii Pyr-1

PhtAc codes for phthalate dioxygenase ferredoxin subunit. It is a subunit of ring-hydroxylating oxygenase enzyme complexes which participate in biodegradation of polycyclic aromatic hydrocarbons. [1] This part encodes for the same protein as BBa_J73042. It only differs in synonym codon usage.

Sequence and Features

Introduction

In this project, we aimed to establish a pyrene degradation pathway in Pseudomonas vancouverensis and to integrate it with the organism's native phenanthrene degradation pathway [2]. It is established that PhtAcAd, as electron transfer components, along with a ring-hydroxylating dioxygenase system formed by pdoA2 and pdoB2, aggregate to create a complex exhibiting dioxygenase activity [3, 4, 5]. This complex is capable of converting Phenanthrene-4-carboxylate, an intermediate product of pyrene degradation, into cis-3,4-dihydroxyphenanthrene an intermediate in the native phenanthrene degradation pathway of P. vancouverensis, thereby facilitating the conjunction of the two pathways [4, 5]. Given that this enzyme complex serves as a critical junction within the pathways, we opted to conduct a more detailed characterization. The components of the electron transport chain, PhtAc a ferredoxin reductase and ferredoxin 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

Enzymes coding sequences were cloned in pQE bacterial expression plasmid with a cleavable N-terminal, 6x-His 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 and for most of the enzymes we were able to get a high yield.

Characterization of ferredoxin reductase PhtAd and ferredoxin PhtAc

The characterization of PhtAd (insert part) activity was performed by evaluating its ability to reduce 2,6-dichlorophenolindophenol (DCPIP), which serves as an electron acceptor [6]. In its oxidized form, DCPIP displays a blue color, which transitions to colorless upon reduction, facilitating the evaluation of PhtAd activity [6]. The decrease in absorbance was monitored at 600 nm [6].

The activity of PhtAc was assessed using a coupled assay. The increase in absorbance resulting from an electron transfer to Cytochrome c signifies an interaction between ferredoxin PhtAc and ferredoxin reductase PhtAd [6]. The increase in absorbance was monitored at 5500 nm [6]. Both reactions needed NADH and FADH as cofactors [6].

Kinetic characterization of PhtAc

Method

Initial measurements were conducted in accordance with the methodology outlined by Wu et al. (2019). 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. (2019), subsequent assays were performed using a PhtAd to PhtAc ratio of 1:3.5. 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 Fig. 3A). 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 Fig. 3C) 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 Fig. 3B) in the first 26 minutes. Conversely, the assay with 100 µM NADH (See Fig. 3D) 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.

Fig. 5 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 the kinetic parameters the Assay was runned with different NADH concentrations ranging from 40 µM to 400 µM. Data obtained from these assays were then analyzed using Michaelis-Menten kinetics (See Fig. 6) and a Lineweaver-Burk plot (See Fig. 7) was constructed to provide a linear representation of the enzyme kinetics to determine key parameters such as Vmax and Km.

Fig. 6 Michaelis-Menten Curve for PhtAc 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.

Fig. 7 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 (Wu et al., 2019) were observed; however, the reaction was conducted with a substantially lower concentration of Cytochrome C and elevated concentrations of enzymes, as the enzymes produced exhibited reduced activity.

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 [3, 4, 5]. 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, High-Performance Liquid Chromatography (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.

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. Furthermore, to eliminate potential statistical error in the assessment of our results, we employed triplicate measurements for each sample and calculated their averages. The results are presented in Figure 1. 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.

Fig. 8 Comparative Analysis of Phenanthrene-4-carboxylat (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 aggregation (a.A) or before aggregation (b.A).

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.

Degradation of Phenanthrene-4-carboxylat by enzyme complex with dioxygenase activity using NADH as a cofactor

References

[1] 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. [2] Y. Yang, R. F. Chen, and M. P. Shiaris, "Metabolism of naphthalene, fluorene, and phenanthrene: preliminary characterization of a cloned gene cluster from Pseudomonas putida NCIB 9816," Journal of bacteriology, vol. 176, no. 8, pp. 2158–2164, 1994, doi 10.1128/jb.176.8.2158-2164.1994. [3] Pagnout, C., Frache, G., Poupin, P., Maunit, B., Muller, J., & Férard, J. (2007). Isolation and characterization of a gene cluster involved in PAH degradation in Mycobacterium sp. strain SNP11: Expression in Mycobacterium smegmatis mc2155. Research in Microbiology, 158(2), 175–186. https://doi.org/10.1016/j.resmic.2006.11.002 [4] Yuan, K., Xie, X., Wang, X., Lin, L., Yang, L., Luan, T., & Chen, B. (2018). Transcriptional response of Mycobacterium sp. strain A1-PYR to multiple polycyclic aromatic hydrocarbon contaminations. Environmental Pollution, 243, 824–832. https://doi.org/10.1016/j.envpol.2018.09.001 [5] Krivobok, S., Kuony, S., Meyer, C., Louwagie, M., Willison, J. C., & Jouanneau, Y. (2003). Identification of Pyrene-Induced Proteins in Mycobacterium sp. Strain 6PY1: Evidence for Two Ring-Hydroxylating Dioxygenases. Journal of Bacteriology, 185(13), 3828–3841. https://doi.org/10.1128/jb.185.13.3828-3841.2003 [6] Wu, Y., Xu, Y., & Zhou, N. (2019). A newly defined dioxygenase system from Mycobacterium vanbaalenii PYR-1 endowed with an enhanced activity of dihydroxylation of high-molecular-weight polyaromatic hydrocarbons. Frontiers of Environmental Science & Engineering, 14(1). https://doi.org/10.1007/s11783-019-1193-5