Jul 2026· Journal of Agricultural and Food Chemistry· 0 citations· 28 references
Medicine
TL;DR
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.
Abstract
The deacetylation of N-acetylglucosamine (GlcNAc) to glucosamine (GlcN) is a key step in enzymatic chitin valorization. We identified a novel GlcNAc deacetylase (AbNGD) from Acinetobacter baumannii with high substrate specificity and catalytic activity but poor thermal stability (half-life of 5.5 h at 37 °C). Using the FireProt platform, we generated a combinatorial mutant, AbNGD-M3 (T93P-C153T-A244M). Its optimal temperature increased to 37 °C, and its half-life reached 28.1 h (5.1-fold improvement), while retaining 97% of wild-type catalytic efficiency (kcat/Km = 3.4 mM-1·s-1). Site-directed mutagenesis confirmed Asp54 and His160 as key catalytic residues. 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. These distal mutations collectively balance stability and activity. Substrate channel analysis identified a likely transport pathway centered on T1. This study expands the deacetylase repertoire and provides a framework for engineering stable industrial enzymes.
: 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
Mannans are abundant plant hemicelluloses, and endo-β-mannanases are important biocatalysts for their conversion into functional manno-oligosaccharides. Here, we report the structural and functional characterization of a glycoside hydrolase family 134 β-mannanase from Aspergillus nidulans (AnGH134) and a structure-guided engineering strategy to improve its performance on locust bean gum. The 1.75 Å crystal structure reveals the conserved lysozyme-like fold of GH134 enzymes and supports an inverting catalytic mechanism with Glu43 and Asp55 as the putative catalytic residues. Docking, mutational, and molecular dynamics analyses indicate that AnGH134 uses an extended substrate-binding groove and that groove-exit residues and the C-terminal region contribute to productive catalysis. Guided by these findings, N-terminal fusion of CBM10 enhanced catalytic efficiency and thermal stability, whereas C-terminal fusion was detrimental. These results provide a framework for engineering GH134 mannanases.
Sheng-Chia Chen, Po-Chih Kuo, Wen-Ming Chen et al.· Journal of Agricultural and...· 0 citations
The rational engineering of an α-L-rhamnosidase (DthRha) to address limitations and enhance its performance for flavonoid production highlights the R783A mutant as a robust and thermally stable biocatalyst with great potential for the sustainable production of bioactive flavonoids.
Haoyu Jia, Luran Wang, Tong Yan et al.· International Journal of Bio...· 1 citation
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.
Teppei Niide, Keita Miyawaki, Hyuga Miyamoto et al.· ACS Chemical Biology· 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
Bidirectional acetylation and deacetylation of small molecules by acetyltransferases (ATs) remain poorly understood. In this study, we present the structural, functional, and computational characterization of a chloramphenicol O-AT from Bacillus sp. PAMC22265 (AT65) that exhibits bidirectional catalytic activity toward the regiospecific C21-acetylation and deacetylation of steroid substrates. The crystal structure of AT65 was determined at 2.40 Å resolution (PDB ID: 24ZY), revealing a trimeric architecture in which the putative active-sites located at the interfaces between adjacent subunits. In vitro kinetic analyses of both acetylation and deacetylation reactions demonstrated substrate-dependent catalytic efficiencies, with comparable activity under the experimental conditions. Molecular docking, 100 ns molecular dynamics simulations, hydrogen-bond analysis, and MM-PBSA free-energy calculations supported stable substrate binding within the active site and suggested that His187 and Asp191 may contribute to substrate recognition and catalysis. Consistent with this observation, substitution of His187 with alanine abolished detectable enzymatic activity, highlighting its functional importance. Whole-cell biotransformation further demonstrated the selective production of C21-acetylated steroid derivatives, some of which exhibited preliminary antiproliferative activity against murine breast cancer cell lines. Collectively, these findings provide structural, biochemical, and computational insights into the bidirectional catalytic properties of AT65 and establish a foundation for further mechanistic studies and the development of selective enzymatic strategies for steroid modification.
Kamal Prasad Regmi, S. Park, Prakash Paudel et al.· International Journal of Bio...· 0 citations
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