Jul 2026· Journal of Agricultural and Food Chemistry· Vol 74, pp. 22088-22097· 0 citations· 43 references
Medicine
TL;DR
A bidirectional loop-engineering strategy is employed to decipher the function of a conserved loop (XDXXP) in GH46 chitosanases and reveals that the loop modulates the geometry, electrostatics, and hydrophilicity of the substrate-binding cleft, thereby influencing substrate recognition and product specificity.
Abstract
Chitooligosaccharides (COSs) are functional oligosaccharides whose bioactivities depend on their degree of polymerization (DP). Enzymatic production of well-defined COSs remains challenging due to the broad product profiles of most chitosanases. Here, we employed a bidirectional loop-engineering strategy to decipher the function of a conserved loop (XDXXP) in GH46 chitosanases. Deleting this loop from Csn-BJ reduced hydrolytic activity by 5% but shifted the maximal product DP from 2 to 4 (yielding 14.2% ± 1.0% of DP4). Conversely, inserting the corresponding loop segment into Csn-BAC enhanced activity by 36% and increased the maximal product DP from 3 to 4 (yielding 20.2% ± 2.5% of DP4). Structural modeling, molecular docking, and molecular dynamics simulations revealed that the loop modulates the geometry, electrostatics, and hydrophilicity of the substrate-binding cleft, thereby influencing substrate recognition and product specificity. This work provides a promising strategy for tailoring chitosanases to produce high-value, customized COSs for food applications.
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
Ulvan lyases play a key role in marine polysaccharide degradation, but how members of the PL40 family achieve their specific product profiles remains poorly understood. Here, we show that variations in the architecture of the substrate-binding cleft dictate whether these enzymes generate disaccharides or longer oligosaccharides as their dominant products. We focused on JpPL40A, a previously uncharacterized ulvan lyase from the marine bacterium Jejuia pallidilutea, and found that it works best at 40 °C and pH 8.0. Using a combination of homology modeling and site-directed mutagenesis, the catalytic mechanism was elucidated, identifying a functional Tyr/His pair (Y260/H429) with R459 responsible for neutralizing the substrate's carboxyl group. Notably, comparative structural analysis revealed that a unique loop region adjacent to substrate binding subsites +5 and +6 dictates its product profile, leading to the predominant generation of unsaturated hexasaccharides. This loop's crucial role was confirmed via truncation mutagenesis, which shifted the main product to disaccharides, and further validated by the discovery of a homologous lyase sharing this structural feature. These results reveal a new structural feature that controls product specificity, helping us better understand how PL40 enzymes work and offering a starting point for designing ulvan lyases that make specific products.
Chengcheng Jiang, Chenghao Hu, Jian-Ping Hao et al.· International Journal of Bio...· 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
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.
Polyketide synthases (PKSs) are privileged enzymatic platforms for generating natural products. Yet, efforts to redesign their acyltransferase (AT) domains for incorporation of non‐native extender units remain constrained by an incomplete understanding of the sequence elements that govern specificity. EryAT6 is the methylmalonyl‐CoA‐specific terminal AT domain of the erythromycin PKS (DEBS). Two conserved motifs within EryAT6, the large‐ and small‐subunit motifs (LSM and SSM), suggest that short sequence segments encode key extender unit selectivity, but their modularity has not been systematically explored. Here, we delineate the sequence–function relationships underlying extender unit selection by combining mutagenesis, motif swapping, and functional reconstitution in Ery6TE. Substitution of ten nonconserved residues across the LSM and SSM revealed some positions that strongly influence the incorporation of larger extender units. Triple‐residue combinations exhibited cooperativity, with the T739A/Y744R/S746G mutant increasing the portion of butylmalonyl‐CoA‐derived product by 42‐fold. Motif exchanges showed that both LSM and SSM can reprogram AT selectivity in AT‐swapped Ery6TE chimeras, enabling the formation of butyl‐substituted pyrones even when the parent domain swap was inactive. Together, these results identify compact determinants of extender unit specificity and establish motif‐level engineering as a strategy to access diverse polyketides.
Sydney D Welch, Gavin J. Williams· ChemBioChem· 0 citations
Starch-modifying glycoside hydrolases (GHs) typically operate via a retaining double-displacement mechanism, involving formation of a covalent glycosyl–enzyme intermediate. This intermediate can be resolved either by water, resulting in hydrolysis, or by a glucan acceptor, leading to transglucosylation. Many GHs exhibit both catalytic activities, although they are classified according to their predominant reaction. For example, branching enzymes (BEs) catalyse α-(1→4) bond cleavage and α-(1→6) branch formation via transglucosylation, while also exhibiting minor hydrolytic and disproportionation activities that broaden their catalytic repertoire. More recent research indicates that the different catalytic activities of BEs can be interconnected, thereby collectively determining the final α-glucan architecture. This challenges the classical view that the predominant branching activity of BEs is catalysed independently. Moreover, the balance between these coupled activities influences substrate specificity and can broaden the substrate scope to include chemically modified starches. Furthermore, the co-application of BEs with other GHs reveals synergistic interactions between catalytic activities, enabling the generation of α-glucan structures that cannot be produced by any of the enzymes individually. In this perspective paper, and based on recent developments, we argue that the catalytic framework of BEs provides multiple strategies for tailoring diverse α-glucan architectures. This enables modulation of structural features across hierarchical levels, from supramolecular to macromolecular organisation. As a result, BEs represent versatile tools for engineering starch functionality beyond digestibility, extending their potential toward pharmaceutical and non-food applications. Looking ahead, we discuss how enzyme-designed and chemically functionalised α-glucan polymers may emerge as a new class of sustainable materials. These materials could provide biodegradable, water-soluble, and renewable alternatives to petrochemical-derived polymers used in personal and home care products.
Maurice K H Essers, Hans Leemhuis, Johannes H. Bitter et al.· Molecules· 0 citations