Mechanistic insights into modulation of productive substrate accessibility for efficient PET depolymerization

Molecular Origin of the Enhanced PET Degradation Activity of LCC-ICCG Revealed by Computational Modeling

Poly(ethylene terephthalate) (PET) hydrolases have emerged as promising biocatalysts for closed-loop plastic recycling. Among the most efficient enzymes reported to date, LCC-ICCG exhibits exceptional PET-depolymerization performance under industrially relevant conditions. However, the molecular basis for its superior activity relative to engineered PETases such as FAST-PETase and HotPETase remains incompletely understood. Here, we combine microsecond-scale molecular dynamics simulations, quantum mechanical cluster calculations, pre-reaction-state analysis, noncovalent-interaction mapping, and distortion/interaction activation strain analysis to compare LCC-ICCG with FAST-PETase and HotPETase. The simulations show that LCC-ICCG samples catalytically competent pre-reaction-state geometries more frequently, mainly because V212 reshapes the local environment around the scissile ester. This residue relieves steric congestion, supports weak C–H···O guided substrate preorganization, and reinforces both the Asp-His catalytic dyad and the W190-associated pocket architecture. Density functional theory calculations further indicate that this preorganized active site lowers the acylation barrier to 15.5 kcal/mol by reducing substrate distortion and strengthening transition-state interactions. High-temperature simulations show that LCC-ICCG better preserves near-attack geometries at 350 K, linking thermal robustness to sustained catalytic preorganization. Moreover, reciprocal I208V mutations in IsPETase-derived enzymes enrich pre-reaction-state populations, supporting the transferability of the V212-centered design principle. Overall, these results establish pre-reaction-state stabilization as a key determinant of PET-hydrolase efficiency and provide mechanistic design rules for engineering next-generation PET depolymerases.

Structure-Guided Terminal Modifications Enhance the Efficiency of PET Degradation

Poly(ethylene terephthalate) (PET) is a widely used plastic whose persistence and improper disposal pose serious environmental and health risks. In this study, three novel PET hydrolases TbPETase, AbPETase, and AfPETase were identified from Thermoanaerobacterales, Acidimicrobiales, and Actinokineospora fastidiosa, respectively. Among these, TbPETase exhibited the highest enzymatic activity and thermostability. Based on structural analysis, we performed semirational truncations targeting the intrinsically disordered N- and C-terminal regions of TbPETase, generating two improved variants ΔN36 and ΔC4. The double mutant, TbPETaseΔN36/ΔC4, demonstrated a 2.3-fold increase in overall enzymatic activity and a 2.6-fold improvement in catalytic efficiency (kcat/Km) compared to the wild-type enzyme, along with significantly enhanced thermal stability. Molecular dynamics simulations revealed that the removal of flexible terminal regions increased the overall structural rigidity of TbPETaseΔN36/ΔC4. This structural stabilization was associated with the formation of a hydrogen bond at T215 and a π–π stacking interaction at W193. In a 100 mL one-pot reaction system, the combination of TbPETaseΔN36/ΔC4 with an engineered BMHETase variant, BMHETase6M, achieved 81.2% degradation of semicrystalline PET powder at 60 °C over 60 h, yielding terephthalic acid as the major product. These findings demonstrate the potential of TbPETaseΔN36/ΔC4 as a highly efficient and industrially applicable biocatalyst for PET degradation.

Overcoming catalytic barriers to reprogram acyltransferases for bis(2-hydroxyethyl) terephthalate hydrolysis.

The extensive use of polyethylene terephthalate (PET) has resulted in severe environmental pollution and ecological stress. Despite advances in PET recycling, current processes struggle to achieve high product value, as the complete conversion to terephthalic acid remains energetically demanding and economically inefficient. The catalytic promiscuity inherent in natural enzyme evolution holds great promise for providing novel candidates to accelerate PET biodegradation and upcycling. Herein, we report for the first time that the acyltransferase metA from Mycobacterium tuberculosis (MtMetA) catalyzes the conversion of bis(2-hydroxyethyl) terephthalate (BHET), an intermediate of PET hydrolysis, into the high-value monomer mono(2-hydroxyethyl) terephthalate (MHET). Guided by molecular dynamics (MD) simulations, we applied a catalytic barrier-minimization strategy to optimize the active-site environment of MtMetA, yielding engineered variants, notably ΔBarrier2 and ΔBarrier3. Specifically, ΔBarrier2 achieved a 3.1-fold increase in MHET yield, while ΔBarrier3 demonstrated a 3.2-fold enhancement in catalytic efficiency (kcat/KM) relative to the wild-type. The truncated MtMetA variants also exhibited enhanced robustness, showing improved thermostability (3.5-fold increase in residual activity at 60 °C for ΔBarrier3), as well as higher tolerance to metal ions and organic solvents. Specifically, ΔBarrier2 displayed a 7.2-fold increase in product yield in Ca2+-containing systems, while ΔBarrier3 retained 2.3-fold higher residual activity in the presence of 50 % (v/v) isopropanol. MD simulations revealed that an enlarged active pocket and a shortened nucleophilic attack distance synergistically govern the enhanced catalytic activity and robustness. This work expands the enzymatic toolbox for PET recycling and targeted BHET degradation, advancing sustainable plastic waste management through biocatalytic innovations.

Rational design of IsPETase variant toward efficient poly(ethylene terephthalate) ambient degradation.

The accumulation of poly(ethylene terephthalate) (PET) waste in the environment poses a severe ecological threat. While extensive research has focused on high-performance PET degradation by thermophilic enzymes, PET hydrolases are efficient under lower-temperature conditions, which would better align with green and energy-saving demands the energy-efficient centralized treatment of PET waste remains underexplored. Herein, based on our previously engineered mesophilic IsPETaseS121P/D186A, we performed rational design to improve its PET degradation activity at relatively low temperature. Through rational design methods including salt bridge construction and hydrophobic engineering, we obtained effective variant PADFL (IsPETaseS121P/D186A/N246D/Y87F/N233L), demonstrating an 8.37-fold activity of IsPETaseS121P/D186A in PET degradation efficiency (56.52-fold of IsPETase). Molecular dynamics (MD) simulations further revealed stronger PET binding affinity, enhanced hydrogen bonding network, and reduced acylation energy barrier. Overall, this work enhances the degradation activity of the PET hydrolase through energy-based rational design and obtained optimized variant PADFL, offering a promising candidate for future efficient PET degradation under mild temperature conditions.

Mechanism-Guided Synergistic Engineering of Substrate Access and Catalytic Microenvironment in Oxidosqualene Cyclase for Enhanced Amyrin Biosynthesis.

Oxidosqualene cyclases (OSCs) catalyze the cyclization of 2,3-oxidosqualene into diverse triterpenoids, yet their intrinsically low catalytic efficiency restricts biosynthetic productivity. Here, we establish a mechanism-guided synergistic engineering strategy that extends beyond conventional active-site engineering by integrating distal substrate access regulation with catalytic microenvironment optimization to enhance the catalytic performance of CrAS from Catharanthus roseus. Structural modeling and mechanistic analyses revealed a conserved catalytic framework involving carbocation-mediated polycyclization and identified a surface-exposed constriction region that regulates substrate access. Guided by these insights, distal surface engineering of the constriction region was synergistically combined with active pocket optimization. The resulting combinatorial mutant, M3 (L323A/T327K/N565I), exhibited a 95.2% increase in catalytic efficiency and enhanced α-amyrin and β-amyrin by 53.2% and 49.7%, reaching 158 mg/L and 63 mg/L, respectively. Multi-scale analyses combining molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations revealed that the enhanced catalytic performance is attributable to increased flexibility of the substrate access pathway, reinforced electrostatic and cation-π interactions, and reduced reaction energy barriers. Notably, distal mutation T327K improved substrate ingress through dynamic modulation of the protein surface, while N565I optimized the catalytic microenvironment by enhancing hydrophobic packing and stabilizing key intermediates. Overall, our findings establish a generalizable framework for engineering complex cyclases and provide a foundation for the sustainable microbial production of high-value triterpenoids.

Elucidating the half-site reactivity mechanism of Salmonella enterica FraB deglycase using native mass spectrometry