Aug 2026· Biotechnology and Bioengineering· 0 citations· 34 references
Medicine
Abstract
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.
Isobutene is an important platform chemical that is still predominantly produced from petroleum-derived feedstocks. The ferulic acid decarboxylase (Fdc) catalyzed decarboxylation of 3-methylcrotonic acid provides a green and sustainable route to bio-based isobutene. However, the poor activity of native Fdc toward this non-natural substrate severely limits its practical application. Here, we developed an integrated enzyme-engineering strategy that combines sequence-cluster mining with distal-site engineering to improve catalytic performance. The engineered triple-site mutant V132C/Y298F/S484A (3MUT) exhibited 5.3-fold the isobutene production of WT. Coupling this mutant with reaction-process optimization and cofactor engineering increased the isobutene yield to 67.9%, representing the highest reported yield to date. Mechanistic studies revealed that distal-site mutations in 3MUT reshaped the active site by disrupting the M293-mediated hydrogen bond that blocks the substrate to active site, then further promoting favorable hydrogen-bond interactions with the substrate via R183 and E292, respectively. These findings establish an efficient route for sustainable bio-based isobutene production and demonstrate the potential of distal-site engineering for improving UbiD-family decarboxylases.
Ting Feng, Xuanyu Cao, Liran Yang et al.· Bioresource Technology· 0 citations
Mogroside VI (Mog VI) is a rare triterpene glycoside from Siraitia grosvenorii with promising bioactivities. However, its biosynthesis is limited by a single rate-limiting glycosylation step converting mogroside V, catalyzed by the inherently low-activity plant glycosyltransferase UGT73-327-2. In this study, we applied a structure-guided engineering strategy to overcome this catalytic bottleneck. By combining substrate-channel expansion with catalytic pocket remodeling, the double mutant W192F/K206E was generated, showing a 22.2-fold increase in catalytic activity. Molecular dynamics simulations and kinetic analyses indicated that the enhanced performance results from an enlarged substrate-access channel, improved substrate-binding stability, and a more favorable active-site geometry that reduces key catalytic distances. Furthermore, coupling the engineered UGT with Arabidopsis thaliana sucrose synthase enabled an in situ UDP-glucose regeneration system, achieving a Mog VI titer of 5.6 g·L-1 with a 76.8% molar conversion. This work establishes an efficient biocatalytic route for Mog VI production and highlights the potential of structure-based glycosyltransferase engineering for the synthesis of rare natural glycosides.
Dong Guo, Yan Zhang, Xupeng Guo et al.· Journal of Agricultural and...· 0 citations
This study provides a practical strategy for engineering thermostable pectate lyases with improved catalytic performance by developing a multidimensional consensus computational framework integrating sequence conservation, structural dynamics, and thermodynamic prediction to identify functional mutation hotspots in PcPel1834.
Ziqi Hou, Gen Lu, Tong Shu et al.· Journal of Agricultural and...· 0 citations
Zearalenone (ZEN) lactonases are promising biocatalysts for ZEN detoxification, yet the catalytic mechanism underlying ZEN hydrolysis remains poorly understood. Here, we combined structural analysis, quantum-mechanical (QM) calculations, and molecular dynamics (MD) simulations to elucidate the catalytic mechanism and guide enzyme engineering. QM and MD analyses identified a near-attack conformation of the catalytic His245 as essential for proton transfer. Crystal structure analysis revealed that mutations within the active pocket enhanced the local hydrophobic microenvironment, thereby optimizing the reactive conformation through an improved substrate positioning and catalytic residue alignment. Engineering the hydrophobic microenvironment significantly enhanced the activity of ZENM toward multiple substrates. Transfer of the engineered region to another ZEN lactonase, ZHD101, also significantly improved the hydrolytic activity, supporting the potential general applicability of this strategy. Hydrophobic microenvironment engineering might represent a promising strategy for modulating proton transfer and provide a potential framework for improving the activity of ZEN lactonases for food and feed detoxification.
Binbin Ouyang, Zhao Huang, Chenshuo Song et al.· Journal of Agricultural and...· 0 citations
This study achieves simultaneous enhancement of catalytic efficiency and stability of a 1,2-rhamnosyltransferase by a distal mutational engineering strategy and provides a promising biocatalyst for rhamnosylated natural product biosynthesis.
Wenjuan Dai, Chaorong Guo, Hongyan Yang et al.· Journal of Agricultural and...· 0 citations
D-Amino acid oxidase (DAAO) catalyzes the oxidation of D-phosphinothricin (D-PPT) to produce optically pure L-PPT, but low catalytic efficiency limits its industrial application. Here, a synergistic strategy combining geometric remodeling and charge engineering was applied to enhance the oxidation activity of our previously developed stable variant TIF-DAAO (S18T/V7I/Y132F) toward D-PPT. Systematic analysis of the active pocket-guided alanine and neutral hydrophilic (Ser/Thr/Tyr) scanning mutagenesis revealed position-specific steric constraints. Subsequent Arg/His scanning tailored the positively charged microenvironment around D-PPT, leading to a triple mutant ATR (F58A/Q335T/P221R) with improved electrostatic complementarity and spatial fit. ATR showed a 10.7-fold increase in catalytic activity, a 35-fold improvement in substrate affinity, and a 380-fold higher catalytic efficiency (kcat/KM) toward D-PPT. Molecular dynamics simulations provided mechanistic insight. Our results demonstrate that the synergistic optimization of substrate tunnel geometry and the electrostatic microenvironment effectively boosts DAAO activity, offering a rational strategy for engineering industrial biocatalysts.
Mengyu Li, Chenchen Fu, W. Zhuang et al.· Journal of Agricultural and...· 0 citations
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