Aug 2026· ChemBioChem· Vol 27· 0 citations· 85 references
Medicine
Abstract
Formate dehydrogenases represent a highly attractive tool for nicotinamide cofactor regeneration to drive biocatalytic reductions and oxygenations by NADH‐dependent oxidoreductases with formate as a cheap, atom‐economic terminal reductant. However, despite the clear benefits over other recycling systems, native FDHs remain rather unpopular, mainly due to their intrinsic, low specific activity. While the active site architecture is generally highly conserved across many FDHs from very diverse origins, smaller patterns of dissonance in the active site's vicinity can serve as hotspots for the rational design of more effective variants. In this study, we have created a series of Candida boidinii FDH variants incorporating nonconsensus amino acids found in Saccharomyces cerevisiae FDH. A double variant (C23S/F285D) was identified that exhibits a significantly higher specific activity, an overall more desirable pH and temperature profile, and an improved stability profile.
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
The large-scale production of indigo, a widely used textile dye, currently relies on chemical processes that consume non-renewable resources and that generate toxic waste, although biocatalysis is emerging as an environmentally friendly alternative. Here, an unspecific peroxygenase from the fungus Daldinia sp. EC12 (DspUPO-I) was subjected to a directed evolution campaign for activity and expression in yeast. With four mutations in the mature protein, the resulting evolved variant showed 95% regioselectivity for indoxyl formation, the precursor of indigo, along with a striking resistance to oxidative inactivation by hydrogen peroxide. These features enable this biocatalyst to synthesize 0.70 g/L indigo on a semipreparative scale. Molecular dynamics simulations highlighted the particular geometry of the broad heme-access channel as the main driving force for the high selectivity and the oxidative stability of this robust biocatalyst during indigo production.
Alejandro Beltrán-Nogal, Ivan Mateljak, Dianelis T. Monterrey et al.· ACS Sustainable Chemistry &a...· 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
: Glucose dehydrogenase (GDH) from Bacillus megaterium IWG3 is a NAD(P)⁺ -dependent oxidoreductase widely used in biosensing and biocatalytic NADPH regeneration. However, its intrinsic preference for NAD⁺ over NADP⁺ limits its application in NADPH -driven processes. Here, we report a structure-guided rational design to invert the coenzyme specificity of GDH by targeting a single residue within the conserved GXXXGXG motif of the Rossmann fold. Molecular docking and structural analysis identified Thr17 as the key residue forming a hydrogen bond with the 2′ - hydroxyl of NAD⁺, thereby discriminating against the 2′ -phosphate of NADP⁺. Three -point mutants—T17G, T17K, and T17R—were constructed, expressed, and kinetically characterized. The T17G mutation dramatically inverted cofactor preference, increasing the catalytic efficiency ratio (NADP⁺/NAD⁺) from 0.78 (wild -type) to 7.5, driven by a 2.4-fold decrease for NADP⁺ and a 4.6 - fold increase in for NAD⁺. Remarkably, the T17K mutant not only shifted preference toward NADP⁺ (specificity ratio 0.96) but also enhanced turnover numbers for both coenzymes by up to 5.2-fold, achieving c atalytic efficiencies of 6.39 mM⁻¹·s⁻¹ (NAD⁺) and 6.15 mM⁻¹·s⁻¹ (NADP⁺) —the highest among all variants tested. In contrast, the T17R mutation severely impaired NADP⁺ binding ( k m = 97.18 mM) and abolished activity. Structural modeling revealed that glycine c reates space to accommodate the 2′ -phosphate, while lysine establishes a favorable electrostatic interaction with the phosphate group; arginine’s bulky guanidinium group causes steric clash. This study demonstrates that a single, rationally designed mutation at position 17 can simultaneously broaden cofactor specificity and improve catalytic efficiency, with the T17K mutant emerging as a superior biocatalyst for NADPH regeneration. The strategy provides a generalizable framework for engineering cofactor preference in short-chain dehydrogenase/reductase family enzymes.
Y. Shen, Keju Jing· International Journal of Fro...· 0 citations
Laccases are biotechnologically valuable enzymes that oxidize phenolic compounds across multiple industries. Their kinetic parameters Km, kcat, and redox potential (E°)—vary with substrate, origin, sequence, and structure, all of which influence electron transfer efficiency toward the trinuclear copper center. Improving kcat and E° is therefore essential for industrial applications. This review analyzes 143 studies, compiling 244 kcat values for ABTS, 125 for 2,6-dimethoxyphenol (2,6-DMP), and 53 for syringaldazine (SGZ). A high-performing laccase was defined by the upper quartile (Q3) of reported values: kcat ≥ 798, 293, and 140 s−1 for ABTS, 2,6-DMP, and SGZ, respectively. Among 36 mutagenesis studies—classified as rational, semi-rational, or directed evolution—directed evolution combined with rational and semi-rational design yielded the greatest improvements, reaching kcat values up to 1328.8 s−1 for ABTS. While Km data are compiled to assess catalytic efficiency, cross-study analysis reveals no consistent directional trend in substrate affinity among engineered variants. In contrast, kcat shows systematic improvement across diverse systems and substrates, establishing it as the primary performance metric for evaluating laccase engineering outcomes.
Alan Rodríguez-Enríquez, N. Rosas-Murrieta, Eduardo Torres· Catalysts· 0 citations
Tyrosinases are binuclear copper enzymes widely used for the hydroxylation of phenolic compounds, yet most exhibit higher activity towards diphenols than monophenols, limiting their utility for catechol synthesis. However, a tyrosinase from Ralstonia pseudosolanacearum GMI1000 (RsTyr) is unusual in favouring monophenol substrates, making it a promising biocatalyst. Here, we combined molecular docking with kinetic characterisation to explore RsTyr's substrate range across eleven monophenols of industrial relevance. Docking studies using homology and AlphaFold models identified key interactions between substrate side chains and catalytic residues, particularly N228, N232 and P239. Experimental validation confirmed broad substrate acceptance, with highest catalytic efficiency observed for resveratrol and tyrosol. Structural analysis suggests that terminal functional groups and sidechain branching strongly influence activity. These findings provide mechanistic insight into RsTyr's substrate specificity and inform strategies for biocatalytic production of valuable catechols.
James Britton, Fang Zhao, Reeta Davis et al.· FEBS Open Bio· 0 citations
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