Presentation Information
[P01-024]Enhancing the Thermal Stability and Catalytic Efficiency of TPA-Degrading Enzymes: TphA and TphB
○Danwoo Kim1, Mihyeon Ryu2, Hyeoncheol Francis Son2,3,4 (1. School of Biological Sciences and Technology, Graduate School Chonnam National University (Korea), 2. School of Biological Sciences and Technology, Chonnam National University (Korea), 3. Institute of Synthetic Biology for Carbon Neutralization, Chonnam National University (Korea), 4. Institute of Systems Biology and Life Science Informatics, Chonnam National University (Korea))
Keywords:
Biodegradation,Rational protein engineering,Protein structure,Thermal stability,Terephthalic acid
[Purpose]
Polyethylene terephthalate (PET) is a recalcitrant plastic that accumulates in the environment and harms terrestrial and marine ecosystems. Biological degradation using microbial enzymes has emerged as an eco-friendly strategy to address this problem. In PET metabolism, terephthalic acid (TPA), generated by PETase, is further converted through 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate (DCD) to protocatechuic acid (PCA). This downstream pathway is considered a metabolic bottleneck, and TphB, which catalyzes the conversion of DCD to PCA, is regarded as the rate-limiting enzyme. Therefore, this study aims to improve the overall pathway flux by enhancing the thermal stability and catalytic efficiency of the TPA-metabolizing enzymes TphA and TphB. Unlike previous studies that mainly focused on PETase engineering, this work targets the downstream TPA metabolic pathway. Improving TphA and TphB is expected to facilitate PCA production, which is significant because PCA can enter the β-ketoadipate pathway and also serves as a valuable platform chemical for industrial applications.
[Results]
Among the tested enzymes, TphB, a DCD dehydrogenase, showed the highest protein expression level. TphB was scaled up for large-scale culture and purified by Ni–NTA affinity chromatography followed by size exclusion chromatography, yielding 82.0 mg of protein. The purified TphB was concentrated to 82.3 mg/mL and crystallized using the sitting-drop vapor diffusion method, producing crystals within 3 days. These crystals were confirmed as protein crystals by X-ray diffraction analysis. In addition, thermal shift assay of TphB revealed two Tm values at approximately 50°C and 64°C.
[Conclusion]
TphA is a multi-metalloenzyme system composed of TphA1, TphA2, and TphA3, and mixed purification confirmed hetero-complex formation between TphA2 and TphA3. Future work will define the functional unit of TphA by cloning each component into vectors with different selection markers and co-transforming them into E. coli. Based on structural information for TphA and TphB, mutation sites will be selected using AI-based prediction models together with structure-based rational engineering. The designed variants will be iteratively evaluated for protein expression, thermal stability, and enzyme activity to obtain TphA and TphB variants with improved thermostability and catalytic efficiency.
[Consideration]
Based on homologous structural analysis and AlphaFold prediction, TphB is likely to exist as a homodimer. The two Tm values observed in the thermal shift assay may therefore reflect its homodimeric structural feature. The first transition may result from disruption of intermolecular interactions between subunits, whereas the second may correspond to unfolding of the intramolecular structure within each subunit.
Polyethylene terephthalate (PET) is a recalcitrant plastic that accumulates in the environment and harms terrestrial and marine ecosystems. Biological degradation using microbial enzymes has emerged as an eco-friendly strategy to address this problem. In PET metabolism, terephthalic acid (TPA), generated by PETase, is further converted through 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate (DCD) to protocatechuic acid (PCA). This downstream pathway is considered a metabolic bottleneck, and TphB, which catalyzes the conversion of DCD to PCA, is regarded as the rate-limiting enzyme. Therefore, this study aims to improve the overall pathway flux by enhancing the thermal stability and catalytic efficiency of the TPA-metabolizing enzymes TphA and TphB. Unlike previous studies that mainly focused on PETase engineering, this work targets the downstream TPA metabolic pathway. Improving TphA and TphB is expected to facilitate PCA production, which is significant because PCA can enter the β-ketoadipate pathway and also serves as a valuable platform chemical for industrial applications.
[Results]
Among the tested enzymes, TphB, a DCD dehydrogenase, showed the highest protein expression level. TphB was scaled up for large-scale culture and purified by Ni–NTA affinity chromatography followed by size exclusion chromatography, yielding 82.0 mg of protein. The purified TphB was concentrated to 82.3 mg/mL and crystallized using the sitting-drop vapor diffusion method, producing crystals within 3 days. These crystals were confirmed as protein crystals by X-ray diffraction analysis. In addition, thermal shift assay of TphB revealed two Tm values at approximately 50°C and 64°C.
[Conclusion]
TphA is a multi-metalloenzyme system composed of TphA1, TphA2, and TphA3, and mixed purification confirmed hetero-complex formation between TphA2 and TphA3. Future work will define the functional unit of TphA by cloning each component into vectors with different selection markers and co-transforming them into E. coli. Based on structural information for TphA and TphB, mutation sites will be selected using AI-based prediction models together with structure-based rational engineering. The designed variants will be iteratively evaluated for protein expression, thermal stability, and enzyme activity to obtain TphA and TphB variants with improved thermostability and catalytic efficiency.
[Consideration]
Based on homologous structural analysis and AlphaFold prediction, TphB is likely to exist as a homodimer. The two Tm values observed in the thermal shift assay may therefore reflect its homodimeric structural feature. The first transition may result from disruption of intermolecular interactions between subunits, whereas the second may correspond to unfolding of the intramolecular structure within each subunit.
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