Aug 2026· Enzyme and Microbial Technology· Vol 201, pp.
110958
· 0 citations· 45 references
Medicine
Abstract
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.
(R)-3-Aminobutanol is a valuable chiral amino alcohol widely used in the pharmaceutical industry. The amine dehydrogenase (AmDH)-catalyzed synthesis with inorganic ammonia as the amine donor represents one of the most promising routes for its production. However, this approach is limited by the low catalytic efficiency of AmDHs toward the unnatural substrate 4-hydroxy-2-butanone, as well as the high cost associated with coenzyme requirements. To address these issues, a structure-guided engineering strategy integrating loop remodeling with dynamically coupled network analysis was employed to modify an AmDH derived from leucine dehydrogenase (GKGB-AmDH). A pentamutant was obtained with a 7.8-fold increase in catalytic efficiency (kcat/Km) and a 6.8-fold increase in specific activity. Molecular dynamics simulations were performed to elucidate the molecular mechanism underlying the improved catalytic performance. A dual-enzyme co-expression system for GKGB-AmDH-M5 and formate dehydrogenase (FDH) was established for in situ NADH regeneration. As a result, the catalytic efficiency toward 4-hydroxy-2-butanone was enhanced, and the conversion reached 83.8% at a substrate concentration of 300 mM, with an enantiomeric excess (e.e.) of > 99.99%. These results demonstrate the feasibility of this engineering strategy and provide a theoretical basis for the efficient and green biomanufacturing of chiral amino alcohols.
Teng‐Kun Huang, Cen Huang, Du Wu et al.· Biotechnology and Bioenginee...· 0 citations
The specificity and catalytic efficiency of enzymes make them attractive for applications ranging from therapeutics to chemical manufacturing. However, it remains challenging to identify specific structural and dynamic mechanisms by which enzymes achieve their catalytic rate enhancements as well as to re-engineer enzymes to improve their catalytic properties. In earlier work we reported the design and selection of KARI mutants with calculated increases in specific activity (i.e., k cat) relative to wild type (WT) for the isomerization step of one of its native substrates: 2-acetolactate (ACL), which leads to the synthesis of the amino acids valine and leucine. Eight mutants were identified with computed improvements in k cat of up to 4 orders of magnitude. In the current study, we investigate the effects of these same mutations on the isomerization of the other native substrate, 2-aceto-2-hydroxybutyrate (AHB, leading to the synthesis of isoleucine). Paralleling our previous work, we use the computational statistical mechanical method transition interface sampling (TIS) to simulate reaction kinetics and compute reaction rate constants. We find that the mutants selected for increased efficiency on ACL had varied levels of activity on AHB–some enhancing reactivity and others diminishing it–with the range in computed AHB rate constants spanning more than 7 orders of magnitude. Analysis of the simulations for WT-AHB revealed that only some of the structural mechanisms associated with mutants’ improved ACL catalysis were expected to transfer to, and thereby improve, AHB catalysis. For two mutants with significantly lower catalytic efficiency on AHB than WT, further analysis identified unique conformational changes that may explain their low activity on AHB.
Elijah Karvelis, Bruce Tidor· Journal of Physical Chemistr...· 0 citations
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.
Marisa Bickmann, Andrea Rodil, Jan Deska· ChemBioChem· 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
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
L‐alanine dehydrogenase (L‐AlaDH) catalyzes the reversible conversion of pyruvate to L‐alanine, mediated by the NAD+/NADH cofactor pair. In this reaction, reductive amination converts pyruvate into L‐alanine in the presence of NADH. While oxidative deamination is widely documented, the reductive amination activity of L‐AlaDH remains less thoroughly characterized. In this study, we performed a comprehensive characterization of L‐AlaDH from Amycolatopsis sulphurea (AsAlaDH) and an engineered variant, Ser192Ala, developed through rational design. The variant was expressed, purified, and evaluated across both reaction directions. We established optimal pH and temperature profiles and determined kinetic parameters, revealing that the Ser192Ala substitution yielded an approximately two‐fold increase in catalytic efficiency (kcat/KM) compared to the wild‐type enzyme for both amination and deamination. We investigated the influence of metal ions (Zn2+, Fe3+, K+, Na+, Li+, Mg2+, Ca2+) on amination activity using two‐way ANOVA and Dunnett's post‐hoc analysis, and assessed storage stability over two weeks at temperatures ranging from –80°C to 20°C. Structural analysis and molecular dynamics simulations suggested improved substrate and cofactor positioning, along with altered active site flexibility, likely contributed to the enhanced catalytic efficiency. Overall, the superior reductive amination activity of the Ser192Ala variant highlights its potential as a robust biocatalyst for industrial L‐alanine production.
T. Aksu, F. Aktaş, Onur Serçinoğlu et al.· ChemCatChem· 0 citations
