Aug 2026· International Journal of Biological Macromolecules· Vol 381, pp.
154119
· 0 citations· 33 references
Medicine
Abstract
Improving enzyme stability without compromising catalytic activity remains a major challenge in protein engineering. Here, we present a co-evolution-guided strategy to enhance both thermostability and catalytic performance of the flavin-dependent monooxygenase MonCI, an enzyme involved in monensin biosynthesis. By combining sequence covariation analysis with structural filtering, a focused library of 15 single mutants yielded 4 variants with increased stability and activity. Combinatorial assembly generated triple, quadruple and quintuple mutants, with the best-performing quadruple variants exhibiting up to a 10 °C increase in melting temperature, a 2.3-fold increase in specific activity, and a 2.1-fold longer half-life, accompanied by enhanced turnover despite reduced substrate affinity. Crystal structures and molecular dynamics simulations reveal that stabilization arises from strengthened intramolecular networks of hydrogen bonds, salt bridges, and hydrophobic interactions, while epistatic effects limit additive improvements. This work provides mechanistic insight into how co-evolving residues modulate enzyme structure and function, presents a useful co-evolution-guided strategy for enzyme design, and advances MonCI as a promising biocatalyst for asymmetric epoxidation.
Glucose dehydrogenase (GDH) is a key enzyme for NAD(P)H cofactor regeneration in industrial biocatalysis. However, conventional engineering approaches are frequently constrained by limited catalytic efficiency and excessive enzyme loading, which collectively compromise process economics and hinder large-scale deployment. In this study, we performed structure-guided rational design by targeting three functionally discrete domains of GDH, including the substrate-binding region, the cofactor-binding pocket, and the interdomain communication interface. An engineered variant GDH-M6 was constructed, which manifests a 35-fold enhancement in catalytic efficiency relative to the wild-type enzyme. In the biocatalytic synthesis of the pivotal chiral intermediate for R-lipoic acid, GDH-M6 reduced enzyme loading by more than 90% and allowed a doubling of the substrate concentration. As a result, overall reaction productivity was substantially increased and the GDH-M6 outperformed wild-type GDH as well as all previously reported mutants under comparable conditions. Notably, the domain-engineering paradigm established herein provides a broadly applicable toolkit for augmenting the catalytic performance of dehydrogenases, and offers a structural blueprint for resolving analogous kinetic bottlenecks that commonly arise in NAD(P)⁺-dependent enzymes utilized for industrial cofactor regeneration.
Min Cao, Xin Hao, Mingjian Zhu et al.· Enzyme and Microbial Technol...· 0 citations
Rhamnosyltransferases are remarkable biocatalysts for the synthesis of rhamnosylated natural products with valuable physicochemical properties and bioactivities. However, their application is hindered by poor stability and low catalytic efficiency. Here, we achieved simultaneous enhancement of catalytic efficiency and stability of a 1,2-rhamnosyltransferase by a distal mutational engineering strategy. The variant M9 exhibited a 589.43-fold extension in half-life, a 2.5 °C increase in Tm, a 13.6 °C increase in T50, and an 8- to 763-fold increase in activity toward diverse flavonoids compared with the wild type. Molecular dynamics simulations provided insights into enhanced thermostability and catalytic efficiency. To demonstrate its synthetic utility, a whole-cell biocatalytic system was constructed in E. coli by coexpressing M9 and UDP-rhamnose synthase, enabling a neohesperidin titer of 1.14 g L–1 without exogenous sugar donor supplementation. This study presents a practical enzyme engineering strategy for simultaneous activity–stability enhancement in glycosyltransferases and provides a promising biocatalyst for rhamnosylated natural product biosynthesis.
Wenjuan Dai, Chaorong Guo, Hongyan Yang et al.· Journal of Agricultural and...· 0 citations
Uricase with improved acid stability is desirable for biomedical and biotechnological applications, yet enhancing the intrinsic acid tolerance of the enzyme while maintaining high catalytic activity remains a challenge. Here, we employed an integrated rational design strategy combining surface charge optimization and B-factor-guided engineering, followed by iterative combinatorial mutagenesis, to engineer urate oxidase from Aspergillus flavus. The final combinatorial variants retained 67% and 64% of their initial activity after 60 min of incubation at pH 4.5, respectively-substantially higher than the 25% retained by the wild-type enzyme-while also exhibiting enhanced specific activities. Mechanistic analyses combining biophysical characterization, molecular dynamics simulations, residue interaction network analysis, and electrostatic calculations suggested that the enhanced acid tolerance may be associated with surface charge redistribution and strengthened van der Waals interaction networks, which may help alleviate electrostatic-repulsion-driven conformational changes and contribute to conformational stabilization. The enhanced catalytic performance may be linked to improved substrate binding and restructuring of the substrate channel. These variants and the underlying design logic illustrate a practical approach to engineering acid-resistant uricase and other pH-sensitive oligomeric enzymes.
Yu-Yue Li, Dan-Yao Zhou, Qi Wen et al.· International Journal of Bio...· 0 citations
d-Allulose 3-epimerase (DAEase) catalyzes d-fructose conversion to d-allulose, but the poor thermostability of Clostridium cellulolyticum H10 DAEase limits its industrial application. Here, we enhanced DAEase thermostability by targeting the subunit interface using PROSS-guided combinatorial engineering and spatial clustering. Candidate mutations were classified into interface core, interface-adjacent, and distal regions, followed by stepwise iterative combination. Two mutants, M5 and M6, retained WT-like activity but showed markedly improved thermostability. The Tm values of M5 and M6 increased by 11.4 and 12.4 °C, respectively, while their half-lives at 65 °C increased 3-fold and 12-fold. Structural analysis indicated that interface mutations promoted salt-bridge reconstruction, distal mutations stabilized monomers, and interface-adjacent mutations optimized the assembly microenvironment. This spatially coordinated strategy provides an effective approach for engineering thermostable multimeric enzymes.
Kaifan Qiu, Xingfei Li, Yuxiang Bai et al.· Journal of Agricultural and...· 0 citations
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
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.
Yangyang Li, K. Jin, Jiangong Lu et al.· Biotechnology and Bioenginee...· 0 citations