Jul 2026· ACS Chemical Biology· Vol 21 8, pp.
2032-2039
· 0 citations· 45 references
Medicine
Abstract
Fe(II)/α-ketoglutarate (αKG)-dependent halogenases that catalyze site-selective C-H halogenation of free substrates without carrier proteins are attractive biocatalysts for diversifying pharmaceuticals and agrochemicals. However, their application remains limited by the narrow diversity of natural halogenases, poor stability, and restricted substrate scope. Protein stabilization is a common strategy to enhance mutational tolerance during enzyme engineering; however, Fe(II)/αKG-dependent halogenases are structurally closely related to hydroxylases, and consensus-based stabilizing mutations risk shifting activity toward competing hydroxylation. To address this challenge, a workflow was designed to improve structural stability while preserving substrate and product specificity by combining computational identification of substrate-recognition residues with Rosetta-based stabilization. This approach was applied to the l-lysine 4-chlorinase BesD from Streptomyces cattleya as a model enzyme. The resulting variants exhibited a T50 increase of more than 45 °C with no loss of substrate specificity or regioselective chlorination activity and served as stable seed enzymes for subsequent substrate scope expansion. This strategy, which systematically excludes substrate recognition- and/or reaction-selectively related residues from the mutation space to preserve native enzyme function, may provide a versatile platform for stabilizing enzymes without substantially compromising catalytic activity.
Deoxynivalenol (DON), a trichothecene mycotoxin commonly found in cereal grains and their derived products, poses significant risks to human and animal health. In previous work, a fusion enzyme composed of the dehydrogenase DADH and the aldo-keto reductase AKR13B3 was engineered to convert DON into the non-toxic 3-epi-DON in a single step. However, the poor thermal stability of this fusion enzyme limited its industrial application. In this study, EVcouplings and the GRAPE-WEB platform were utilized to identify key amino acid residues governing the thermal stability of the fusion enzyme AKR13B3–DADH. Through single-point mutation screening and the combination of beneficial mutation sites, a triple mutant M361L/T508Y/Y603F (M1) was obtained. The half-life of M1 at 50 °C reached about 500 min, representing a 16.4-fold increase compared with the wild type, while its catalytic activity increased by 2.7-fold. The apparent melting temperature increased by approximately 4 °C. Molecular dynamics simulations verified that the improved thermostability results from reduced conformational flexibility in key regions, enhanced structural packing, and a strengthened hydrogen bond network. These results demonstrate the successful development of a thermostable DON-detoxifying fusion enzyme and provide a practical basis for its industrial application.
Yi-Ting Pan, Hao Zhu, Qing-Wei Jiang et al.· International Journal of Mol...· 0 citations
Gallic acid is a bioactive polyphenol with poor solubility and stability. Enzymatic α-glucosylation can address these limitations, but regioselective synthesis among multiple phenolic hydroxyls remains challenging. Here, we engineer a sucrose phosphorylase from Bifidobacterium adolescentis (BaSP) for the switchable synthesis of gallic acid 4-O- and 3-O-α-d-glucopyranosides. Structure-guided rational design identifies four key regions controlling substrate orientation. The double mutant I231A/D400E achieves >90% selectivity for the 4-O isomer with 82% yield in 2 h. Strikingly, the mutant L341 V/T157G completely reverses regioselectivity, producing the 3-O isomer with >98% yield and >99% selectivity. Molecular docking reveals that these mutations remodel the active pocket and substrate channel, positioning the target hydroxyl group adjacent to the catalytic center. The resulting α-glucosides show up to 12-fold improved water solubility and superior cytoprotective, anti-inflammatory (IL-1β), and antioxidant (HO-1/NQO1) activities compared to gallic acid. This work provides two high-performance, regiocomplementary biocatalysts for green synthesis of pharmaceutically relevant gallic acid glycosides and offers mechanistic insights into regioselectivity control in sucrose phosphorylases.
α-Ketoglutarate (α-KG)-dependent nonheme iron enzymes catalyze a diverse array of oxidative transformations essential for natural product biosynthesis. However, the mechanism by which α-KG/Fe-dependent halogenases achieve selective halogenation while circumventing the thermodynamically favored hydroxylation pathway remains a subject of intense debate. In this study, we elucidate the halogenation mechanism in SyrB2 through extensive computational and crystallographic investigations. Our work reveals that metal coordination dynamics plays a pivotal role in controlling selective C─H bond activation and chlorination in SyrB2. The transformation of the Fe(IV)-oxo species from an equatorial to an axial conformation enables hydrogen atom transfer from the substrate C─H bond. Subsequent re-isomerization of the Fe(III)-OH intermediate to the equatorial conformation is critical for promoting selective chlorination while minimizing competitive hydroxylation. The proposed mechanism is supported by multiple experimental observations, including Mössbauer spectroscopy, nuclear resonance vibrational spectroscopy (NRVS), 2H-HYSCORE spectroscopy, and kinetic analysis of reactions with various substrates.
Wenli Yuan, Jia-Yong Huang, Jia Liu et al.· Angewandte Chemie· 0 citations
The deacetylase repertoire is expanded and provides a framework for engineering stable industrial enzymes and molecular dynamics simulations revealed that T93P reduces backbone flexibility, C153T enhances β-sheet rigidity via hydrogen bonding, and A244M improves hydrophobic packing by filling a core cavity.
Zechang Sun, Yuxin Xia, Yiran Li et al.· Journal of Agricultural and...· 0 citations
Nonheme iron/α-ketoglutarate-dependent dioxygenases (Fe/αKGDs) catalyze diverse oxidative transformations, including hydroxylation, desaturation, and ring expansion. Although these reactions are well established, Fe/αKGD-catalyzed epimerization remains rare and has not been characterized in ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we report the RiPP-modifying enzyme YmI, a bifunctional Fe/αKGD that catalyzes Cβ-hydroxylation of Phe19 and Cα-epimerization of Ile20 on the precursor peptide YmA toward the formation of YM-216391. The hydroxylation generates (2S,3R)-β-hydroxyphenylalanine, whose l-threonine-like stereochemistry facilitates subsequent formation of an unusual 5-phenyloxazole moiety. Substrate profiling analyses reveal leader peptide dependence for catalytic activity. Structural and docking studies provide insights into the substrate binding and active site architecture. Deuterium labeling demonstrates that YmI catalyzes Ile epimerization by directly abstracting the Cα-hydrogen, generating a Cα radical that is subsequently reprotonated by a nonexchangeable hydrogen source within the enzyme. These results unveil an unprecedented mechanism and novel bifunctionality, markedly expanding the reaction landscape for αKG-dependent enzymes in RiPP biosynthesis.
Unknown authors· Journal of the American Chem...· 0 citations
BbAS is established as a thermostable and industrially promising biocatalyst for efficient turanose production through sequence-based analysis and molecular dynamics simulations.
Jeon-Uk Kang, Ye-Jin Kim, Dong-Ho Seo et al.· Journal of Agricultural and...· 0 citations
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