Over the past decade, an increasing number of functional microbial-like terpene synthases (MTPSLs) have been reported in non-seed plants. However, whether the traditional Chinese medicinal plant H. serrata harbors such enzymes and their corresponding functions remains unexplored. In this study, we mined the transcriptome of H. serrata and identified a microbial-like terpene synthase, HsMTPSL1, which produces multiple diterpene products. Following isolation and structural elucidation, seven distinct compounds were obtained, representing three skeletal types: spatane, prenylkelsoene-type, and biflorane. Among these, compound 7 is a novel biflorane diterpene. Structural analysis and subsequent mutagenesis revealed critical residues governing the formation of distinct skeletons, uncovering the multifunctional nature of this enzyme. Notably, the S224A mutation significantly enhanced the production of spatane diterpene compound 1 by 11.6-fold, demonstrating the potential for protein engineering to improve the yield of this bioactive marine-specific diterpene. Transcriptomic profiling revealed that HsMTPSL1 is highly expressed in sporangia, and co-expression analysis with cytochrome P450s identified the CYP781 subfamily as candidates potentially involved in the downstream modification of these skeletons. Collectively, we report the first MTPSL from H. serrata and characterize it as a multifunctional diterpene synthase. Through structure-guided mutagenesis, we uncovered the molecular basis of its functional versatility, with the S224A mutation providing a powerful tool for enhancing the yields of all three diterpene skeletons, thereby laying a foundation for future protein engineering and synthetic biology applications.
Background: Malaria parasites import essential nutrients from plasma into their host erythrocytes through the plasmodial surface anion channel (PSAC), a conserved ion and nutrient channel on the infected cell surface. A parasite-encoded ternary complex consisting of CLAG3, RhopH2, and RhopH3 determines PSAC activity, but the precise contributions of each member to formation of the nutrient uptake pore remains uncertain. Methods: Here, we devised a two-step CRIPSR transfection strategy to examine an amphipathic CLAG3 helix, termed α-helix 44 (α-H44), as a candidate pore-lining domain. Results: A CLAG3 truncation protein without α-H44 phenocopies a CLAG3 knockout line, suggesting a critical role of α-H44 in formation of the nutrient channel; CLAG3 restoration using a recodonized α-H44 restores PSAC activity fully. A saturation mutagenesis library that splits the helix into four sequential segments was devised and implemented. Two engineered mutants exhibit distinct PSAC phenotypes; their cultures failed to expand in a modified medium that approximates in vivo nutrient availability. Conclusions: These studies support a α-H44 role in channel permeation and block by a strain-specific inhibitor. Our strategy will enable saturation mutagenesis to determine how PSAC achieves its unique ion and nutrient selectivity and should help guide drug discovery against this antimalarial target.
The genome of every cancer cell carries a record of the mutational processes that have acted throughout its history. Mutational signature analysis, which infers the activity of mutagenic processes from their characteristic base-change patterns, has become an indispensable tool for interpreting somatic mutations. However, this framework captures only which types of mutations a process generates and not where in the genome they occur - a distribution influenced by replication timing, chromatin organization, transcription, DNA secondary structure, and other genomic features. Here, we present a generative probabilistic framework (MuTopia) that jointly infers mutational spectra and their genome-wide topography as nonlinear functions of genomic and epigenomic state. Applied to whole-genome sequencing data from 15 tumor types, MuTopia reveals that mutational processes fall into eight conserved topographic archetypes, or topotypes, shaped primarily by replication timing and chromatin state. Diverse mutational processes converge upon this limited repertoire, indicating that the genomic distribution of mutagenesis is constrained less by the source of damage than by how that damage is processed. Individual mutational processes exhibit state-dependent variation in their genomic distributions: the same signature can adopt distinct topotypes depending on repair proficiency and replication stress. For instance, SBS8 shifts from a canonical late-replicating profile in homologous recombination-proficient tumors to an early-replicating, stress-associated topotype in HR-deficient tumors, and replication stress similarly reshapes the genomic distribution of APOBEC editing. Topotypes, therefore, provide a classification of mutagenesis distinct from spectral signatures, capturing aspects of tumor biology that spectra alone cannot resolve.
Density-dependent population regulation is widespread in the animal kingdom, but the underlying molecular mechanisms remain poorly understood. Here, we show that C. elegans animals respond to crowding stress by secreting CPR-4, a homologue of human cathepsin B cysteine protease, leading to chromosomal DNA damage in germ cells and high density-induced deficiencies that include increased embryonic lethality and larval arrest and decreased brood size. CPR-4 mediates these crowding responses through the insulin-like growth factor receptor DAF-2, multiple components in the insulin signaling pathway, and the SKN-1/Nrf transcription factor. Whole genome sequencing analyses of animals from 10 generations of continual growth in the crowded condition reveal that CPR-4-induced DNA damage produces an average of 2.8 more de novo genome mutations per animal per generation and a 87% increase in mutation rate compared with animals grown in the uncrowded condition. CPR-4-induced mutagenesis also facilitates evolution of the genomes through multi-generational crowding selection, leading to biased mutation distributions towards the intergenic regions over the gene bodies and crowd-dependent growth advantage. Therefore, CPR-4 acts as a crucial crowd-responding factor to induce genome mutagenesis, driving genome evolution and competitive growth of animals in response to crowding stress.
Heparinase III (Hep-III) plays an important role in the degradation of heparan sulfate and heparin, but its instability limits its applications. Herein, a distal mutagenesis strategy based on sequence conservation analysis and molecular dynamics (MD) simulations was employed to enhance the thermostability of Hep-III from Pedobacter schmidteae (PsHep-III). The V183A/D378G mutant (M2a) exhibited a 31.21-fold increase in half-life (t1/2) at 35 °C relative to the wild type (WT). Furthermore, the melting temperature (Tm) and the temperature at which 50% residual activity was retained after 1 min (T501) of M2a were increased by 23.66 and 8.02 °C, respectively. The specific activity of M2a was 104.16% relative to that of the WT. Analysis of intramolecular interactions, protein surface charge and MD simulations indicated that enhanced local rigidity and increased surface potential were the main contributors to the overall stability improvement of M2a. This study provides an effective strategy for improving the thermostability of Hep-III.
Bacterial β-barrel pore-forming toxins, including Staphylococcus aureus α-toxin (Hla) and Bacillus cereus toxins hemolysin II (HlyII) and cytolytic toxin K2 (CytK-2), are secreted by bacterial cells as water-soluble monomers. These monomers assemble within lipid bilayers to form cylindrical pores, leading to lysis of target eukaryotic cells. We created mutant forms of these toxins that, based on the results of X-ray structural analysis of Hla and the prediction of the 3D structure of HlyII and CytK2, can form intramolecular disulfide bonds in monomers. The substitutions were made in the region responsible for toxin insertion into the target membrane. The mutant forms reversibly altered their hemolytic activity depending on the presence of reducing reagents and were non-toxic when injected into experimental animals. The immune response to injection of the mutant forms of Hla and CytK-2 toxins resulted in higher antibody titers against the wild-type toxins and a higher level of immunological memory than with injection of the HlyII mutant. The mutant form of CytK-2 demonstrates the properties of a prototype vaccine, as immunization with this protein protects animals against the effects of the wild-type toxin.
Alternative DNA structure-forming (i.e., non-B) sequences such as H-DNA-forming sequences are enriched at chromosomal translocation hotspots in human cancer genomes, underscoring their role in genomic instability. H-DNA is particularly susceptible to DNA damage by reactive oxygen species (ROS), a common byproduct from both endogenous metabolism and environmental contaminants, thereby exacerbating its mutagenic potential. Oxidative lesions within B-DNA are efficiently processed by base excision repair (BER), whereas H-DNA is processed in a mutagenic fashion by nucleotide excision repair (NER). Thus, we speculate that the repair of oxidative lesions within H-DNA will promote aberrant BER and NER processing, ultimately enhancing mutagenesis. Here, we examine the processing of oxidative damage within H-DNA by measuring the changes in mutation frequencies and spectra, as well as the association with key NER and BER proteins in human cells in the presence or absence of specific DNA repair proteins. Our results demonstrate that oxidatively damaged H-DNA serves as a substrate for both BER and NER and reveals an interplay between BER and NER proteins, which influences mutation outcomes. This novel framework establishes a link between oxidative stress, DNA repair, and H-DNA-associated mutagenesis, providing insight into how environmentally relevant DNA damage can drive sequence-specific genomic instability at cancer-associated hotspots.
We describe the identification and characterization of the HMB-3 variant, produced by a P. asiatica isolate from a patient in Switzerland. A carbapenem-resistant P. asiatica isolate was sent to the Swiss National Reference Center for Emerging Antibiotic Resistance for investigation. Antibiotic susceptibility testing was performed according to CLSI guidelines. WGS was performed on Illumina and Oxford Nanopore platforms. The blaHMB alleles were cloned into the pCR-Blunt II-TOPO plasmid. Site-directed mutagenesis was performed on blaHMB-1 and blaHMB-3 inserted into the pTOPO plasmids. Purified HMB-1, HMB-3, NDM-1 and IMP-1 were used for steady state kinetic measurements of hydrolysis of selected beta-lactams. The isolate was obtained from a tracheostomy wound swab. Susceptibility testing showed that it was resistant to all beta-lactams, except aztreonam; however, no classical carbapenemase genes were identified by routine testing. WGS produced a complete chromosome of 6.1 Mb but no plasmids, and identified a gene encoding a novel MBL, namely HMB-3, differing from HMB-1 by 23 amino acid substitutions. HMB-3 was chromosomally encoded and located within a 25.6 kb genomic island. HMB-3 conferred resistance to most beta-lactam antibiotics, except piperacillin (MIC 4 mg/L), aztreonam (0.125 mg/L) and cefiderocol (2 mg/L). Site-directed mutagenesis of blaHMB-1 and blaHMB-3 revealed that a single amino acid change, E181H/H181E, in active site loop 10, could significantly alter the MIC of cefiderocol. HMB-3 demonstrated increased hydrolytic activity against cefiderocol compared with HMB-1 and NDM-1. Here we described a novel MBL enzyme responsible for acquired resistance to carbapenems and reduced susceptibility to cefiderocol in Pseudomonas spp.
Wheat starch composition and content significantly influence the processing and end-use quality of flour. In this study, we characterized a novel wheat mutant (M393) generated via γ-ray mutagenesis, which exhibited altered starch content and superior bread-making performance compared to the wild type (WT). Comprehensive comparison analysis shows that the mutant M393 has longer dough stability time, higher tensile strength and better ductility than the wild type, and the volume of bread is larger, the texture of bread is finer, and the taste of bread is better. Microscopic observations, together with laser diffraction particle size analysis, revealed smaller starch granules and an increased proportion of B-type granules in M393.The total starch and amylose content of the mutant M393 were significantly decreased, while the amylopectin content was increased. Transcriptomic analyses revealed downregulated expression of the GBSS gene, which is responsible for amylose synthesis, while genes related to amylopectin synthesis (SBE, SS, and DBE) were upregulated, corresponding to the shifts in starch composition. In addition, a co-expression network of transcription factors and starch synthesis genes was constructed, and new transcription factors such as MYB89, SBP91, bHLH131, HD-ZIP67, and HD-ZIP66 were identified as putative regulators that may be involved in the expression of starch genes. The results of this study provide new insights into the transcriptional regulation of starch biosynthesis and provide a valuable theoretical basis for breeding wheat varieties with better processing quality.
The malarial parasite Plasmodium falciparum cleaves to host haemoglobin through a cascade of proteolytic enzymes. The cysteine protease, Falcipain-2 (FP2), plays an essential role in the process and is important for parasite survival, making it a potential drug target. However, similarities with host cysteine cathepsins hamper selective inhibition, thus necessitating detailed structural and functional characterisations of FP2. This study uncovers a new regulatory role for polyethylene glycol 400 (PEG400) on FP2 activity. PEG400 inhibits FP2 activity on small peptide substrates and azo-casein, while enhancing haemoglobin degradation, exerting a dual effect on FP2 catalysis. Mixed-type inhibition has been observed for PEG400 against small peptide substrates of FP2. This is consistent with the binding of PEG400 to the catalytic cleft, confirmed by fluorescence quenching and docking studies. Unlike typical nonspecific PEG-protein interactions, PEG400 adopts a fit within the catalytic region of FP2 and partially overlaps with leupeptin-binding sites, albeit with a lower affinity. Computational analysis further identifies a previously undescribed allosteric binding pocket of PEG400, which is further supported by in silico mutagenesis and molecular dynamics simulation studies. This pocket exhibits minimal conservation in human cathepsins, suggesting its selective potential. In contrast to this inhibitory role, biochemical assays revealed that PEG400 promotes haemoglobin proteolysis. Spectroscopic analyses further suggest that PEG400 alters haemoglobin structural dynamics to favour proteolysis. Interestingly, ENM-based normal mode analysis revealed that upon haemoglobin binding, PEG400 restricts the FP2 hinge-bending motion, improves FP2-haemoglobin proximity, and PEG400 is simultaneously dislodged from the active site, thereby promoting proteolysis. Altogether, these results reveal a previously undescribed mechanism of FP2 regulation, highlighting new therapeutic avenues.
Gene therapy using recombinant adeno-associated virus (AAV) vectors has emerged as a promising approach for treating genetic disorders, including Leber congenital amaurosis 2 (LCA-2) induced by RPE65 mutation, a severe form of inherited retinal dystrophies (IRDs). This review provides recent advancements, methodological strategies, and therapeutic aims related to AAV vector-mediated retinal gene therapy for LCA-2 induced by RPE65 mutation. The literature search was performed using the PubMed, Scopus, and Web of Science databases, focusing on studies that examine gene therapy as a potential therapeutic strategy for LCA-2 by introducing functional copies of the RPE65 gene into affected cells. Due to their ability to efficiently deliver therapeutic genes without significant immune responses or mutagenesis events, AAV vectors have shown efficacy in restoring retinal and visual functions in animal models of LCA-2. Advancements in molecular biology and retinal surgery have enabled clinical studies and trials for gene therapy in LCA-2, providing a foundation for further research and improving treatment outcomes. While there is currently no known cure for IRDs, treatments such as vitamin supplementation, gene therapy, and assistive devices can help manage symptoms and slow disease progression. Ongoing clinical trials are investigating novel therapies, including stem cell therapy and gene editing technologies, to expand treatment options for IRDs. The favorable safety profile and proven efficacy of AAV vectors, combined with their capacity for sustained transgene expression, position them as ideal vehicles for ocular gene therapy applications. However, immune responses and off-target effects must be addressed carefully.
Programmed cell death 4 (Pdcd4) is a well-established tumor suppressor and inhibitor of protein translation. Although Pdcd4-mediated translational repression contributes to tumor suppression, emerging evidence suggests that Pdcd4 also exerts translation-independent functions. In this study, we found that Pdcd4 suppresses tumorigenesis through direct interaction with the rapamycin-insensitive companion of mTOR (Rictor), a core component of the mTORC2 complex. Using deletion mapping and site-directed mutagenesis, we defined the Rictor-binding domain of Pdcd4 and identified three critical residues, R105, K108, and R110, for this interaction. Co-immunoprecipitation and in vitro kinase assays demonstrated that Pdcd4 binding to Rictor disrupted mTORC2 complex assembly and inhibited its kinase activity. Reverse phase protein array analysis revealed that 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key regulator of glycolysis, was markedly upregulated in Pdcd4-knockdown cells. Restoration of wild-type Pdcd4, but not a Rictor-binding-deficient mutant, reduced PFKFB3 protein abundance by promoting ubiquitin-proteasome-mediated degradation. Functionally, Pdcd4-Rictor interaction suppressed glycolytic activity and inhibited tumor cell proliferation in cultured cells and xenograft models. Consistent with these findings, non-small cell lung cancer (NSCLC) tissues exhibited significantly elevated protein levels of Rictor and PFKFB3 compared with adjacent normal tissues, with a positive correlation between their expression. Collectively, these results demonstrate that the translation-independent mechanism by which Pdcd4 disrupts mTORC2 signaling and downregulates PFKFB3 plays a critical role in suppressing NSCLC growth and glycolysis.
Efficient secretion of heterologous proteases in S. cerevisiae remains a major bottleneck due to host cytotoxicity and secretory stress. In this study, we developed a yeast chassis strain through three rounds of iterative evolution, combining ultraviolet (UV) and atmospheric and room temperature plasma (ARTP) mutagenesis with a fluorescence-based screening pipeline. The final strain, MA10Ah121, exhibited protease production nearly three times that of its parental strain LB184M and maintained genetic stability across passages. Importantly, MA10Ah121 supported enhanced expression of two structurally distinct collagenases, ColG (Class I) and ColH (Class II), demonstrating its broad utility for recombinant protease production. Whole-genome sequencing revealed substantial chromosomal remodeling, including reversion of a 151 kb duplication enriched in genes related to energy metabolism and protein synthesis, along with widespread structural variations. These changes reflect a genome-wide adaptation that rebalances biosynthetic capacity and stress tolerance. This work provides a robust and versatile S. cerevisiae chassis for the biosynthesis of proteases and other challenging recombinant enzymes in industrial biotechnology applications.
The frontotemporal dementia-linked S320F mutation in the microtubule-associated protein tau promotes spontaneous aggregation, yet the structural basis of its amyloidogenesis remains unclear. Using cryo-electron microscopy, we determined the structure of an S320F 295-330 tau fibril composed of parallel chains stabilized by the 306 VQIVYK 311 amyloid motif, with S320F buried in the fibril core and a C322-C322 disulfide linking two protofilaments. Although cysteines are dispensable for fibril formation by isolated peptide fragments in vitro, tau repeat domain constructs containing both C291 and C322 generate more potent seeds in cellular assays. In contrast, the C322S mutation suppresses spontaneous aggregation of S320F tau in cells, and combined C291S and C322S mutations inhibit seeded aggregation in both wild-type and S320F contexts. Systematic alanine mutagenesis coupled with seeding by tauopathy-derived material identifies cysteine residues as critical determinants of tau seeding, comparable in importance to core amyloid motifs. Together, these findings establish cysteines as central chemical regulators of tau aggregation and propagation.
Kasugamine (2,4-diamino-2,3,4,6-tetradeoxy-d-mannose, 1) is a rare multideoxy diamino-sugar forming the core skeleton of the commercial aminoglycoside antibiotic Kasugamycin (KSG, 2), an agricultural fungicide that has been used against rice blast disease for more than 60 years. Despite the reported biosynthetic gene cluster (BGC) for 2, the enzymatic logic leading to the formation of 1 remained unclear. Here, we report the biosynthetic mechanism of 1. Five enzymes─KsgQ, KsgB, KsgD, KsgR, and KsgC─can biochemically convert uridine diphosphate-N-acetyl-d-glucosamine (UDP-GlcNAc, 4) to generate the sugar donor 6, which is incorporated into 2, as confirmed by stable isotope-labeled feeding experiments. KsgB is characterized as the first enzyme catalyzing the deacetylation of uridine diphosphate-N-acetyl-d-mannosamine (UDP-ManNAc, 5), while KsgR represents the first UDP-sugar 3-dehydrase catalyzing the C-3 deoxygenation of the KsgD product (8) via the cofactors pyridoxal-5'-phosphate (PLP) and l-glutamic acid (l-Glu), and employs a ColD-like catalytic mechanism, as shown by crystallographic analysis, molecular docking, and site-directed mutagenesis. Our findings unravel the biosynthetic mysteries of 1, highlight the intriguing strategies for biosynthesizing diamino-sugars in nature, and provide some evidence for further completely uncovering the biosynthetic logic of 2.
Near-infrared fluorescent proteins (niRFPs) are valuable markers for tracking cellular phenomena as they offer greater imaging depth, lower background, and minimal invasiveness relative to other fluorescent probes. The small ultrared fluorescent protein (smURFP) is the brightest niRFP currently reported and has been the subject of several mutagenesis studies to improve its biophysical characteristics. Here, we demonstrate a systematic approach to exploring the mutational landscape of smURFP using a comprehensive deep mutational scanning (DMS) library to identify novel smURFP mutations which confer greater in vivo fluorescence. By observing changes in relative abundance between naïve and fluorescence sorted populations, we provide analysis of the enrichment of all possible single-codon substitutions, insertions, and deletions for smURFP. Enriched populations yielded a series of seven single-codon substitutions which confer a threefold increase in in vivo fluorescence in E. coli when combined. Finally, we assess the potential underlying mechanisms for increased fluorescence by characterizing the biophysical and photophysical properties of the mutant niRFP sequences. We confirm that two of the derived smURFP sequences have a higher molecular brightness than wild type and yield the brightest niRFP reported.
Polyphenolic compounds are widely explored for health benefits, including hypertension, but their active ingredients, molecular targets, and mechanisms remain poorly defined. We identify the xanthone Mangostin from Garcinia mangostana as a potent modulator of several potassium channels, with large-conductance K+ (BK) channels as its primary target for vasorelaxation. Mangostin-activated BK channels as α subunits alone, in complexes with vascular β1 subunits, and in reconstituted BKα/β1-Cav nanodomains. It shifted BK voltage activation to more negative potentials by antagonizing channel closure and promoting channel opening without markedly altering Ca²+ sensitivity. Docking, competition, single-channel analysis, and mutagenesis localized the binding site in the pore cavity below the SF, involving gating-critical S6 residues I308, L312, and A316, and suggest that Mangostin stays bound in closed and open states. These findings establish BK channel activation as the core molecular mechanism driving Mangostin's vascular effects and define its structural mode of action, informing nutraceutical safety assessment and BK-targeted drug design.
In this study, a wild-type alkaline protease-producing Bacillus strain isolated from soil was biochemically and molecularly characterized. The strain was identified as Bacillus subtilis PTK56 based on 16 S rRNA analysis. Random mutagenesis using EMS (Ethyl methanesulfonate) generated multiple variants, and the mutant with the highest protease productivity was selected. Enzyme production conditions for both strains were optimized, and the partially purified enzymes were comparatively characterized. The mutant protease exhibited a 1.34-fold higher activity than the wild type. Zymogram analysis confirmed the functional impact of the mutation through the appearance of an additional activity band. Both enzymes displayed an optimal pH of 9.0 and an optimal temperature of 55 °C. They retained ≥ 97% of their initial activities between 30 and 60 °C and maintained ≥ 95% stability within the pH range of 6.0-11.0. In the presence of metal ions (5-10 mM) and organic solvents (10%), both enzymes preserved more than 90% activity. In non-ionic detergents (1% Triton X-100, Tween-20, and Tween-80), both enzymes exhibited high stability, retaining > 90% activity with Tween-20 and Tween-80, while the wild-type retained ~ 79% and the mutant ~ 92% with Triton X-100. The mutant enzyme showed markedly higher stability, performing 39% better than the wild type in 5% Triton X-100 and 26% better in 5% H₂O₂. Both enzymes also maintained ≥ 80% stability across 1-15% NaCl concentrations. HPLC analysis of casein hydrolysis products revealed higher amino acid release by the mutant enzyme. Overall, both proteases demonstrate strong potential for applications in detergent, photographic, and pharmaceutical industries.
Aflatoxin B1 (AFB1) poses a serious threat to global food and feed safety. Laccase-based enzymatic degradation represents a promising green strategy for AFB1 removal; however, its industrial application is severely limited by the rapid thermal inactivation of wild-type enzymes under high-temperature processing conditions (>70 °C). Here, we engineered the thermal stability of a laccase from Bacillus amyloliquefaciens B10 through an integrated strategy combining computational structural biology with semi-rational design. By coupling molecular dynamics (MD) simulations with folding free-energy (ΔΔG) calculations, we identified key flexible regions associated with thermal instability and subsequently implemented iterative saturation mutagenesis. The best single mutant, R196C, retained more than 96% relative activity after heat treatment at 80 °C for 10 min. Further iterative mutational stacking progressively enhanced thermostability: the R90E/R196C double mutant showed 1.25-fold higher activity at 80 °C than R196C, and the R90E/R196C/H54F triple mutant showed a further 1.16-fold increase over the double mutant. The final quadruple mutant, R90E/R196C/H54F/R253I, achieved 86.9% AFB1 degradation at 80 °C after 24 h. High-temperature MD simulations (100 ns at 353.15 K) indicated that the enhanced thermostability was associated with reduced conformational flexibility, lower radius of gyration (Rg) and solvent-accessible surface area (SASA), and a coil-to-β-sheet transition that contributed to stabilization of the protein core. In addition, efficient secretory expression of the engineered enzyme was achieved in Pichia pastoris, reaching 3.0 U/mL, while the crude enzyme maintained more than 70% activity at 80 °C. Collectively, these results provide a practical basis for the rational engineering and scalable production of thermostable biocatalysts for AFB1 detoxification-related applications of AFB1 control, and offer broader insights into the targeted enhancement of thermal stability in industrial enzymes.
Efficient enzymatic depolymerization of chitin is often limited by the high crystallinity and low accessibility of the native substrates. Here, a semirational protein design was applied to improve the catalytic efficiency of the GH18 Chitinase Chi1 from Chitinibacter sp. GC72 by targeting residues in the substrate-binding channel. Four residues (Lys440, Trp467, Tyr499, and Arg527) were identified as potential functional hotspots and subjected to alanine scanning and site-saturation mutagenesis. Among the resulting variants, mutant Y499C exhibited significantly enhanced catalytic performance, showing 42% and 83% higher specific activities toward colloidal chitin and crystalline chitin, respectively, compared to the wild type. Kinetic analysis revealed that the Vmax and catalytic efficiency (kcat/Km) of Y499C increased to 1.57-fold and 2.29-fold relative to the wild-type enzyme. In addition, Y499C achieved higher conversion than that of the wild type, reaching 29.5% for crystalline chitin and 20.45% for colloidal chitin. Molecular simulations indicated that the Y499C substitution reshaped the substrate-binding channel, stabilizing the enzyme-substrate complex and potentially improving the substrate accessibility within the catalytic cleft. These findings demonstrate that substrate-channel hotspot engineering is an effective strategy for enhancing GH18 chitinases and provide insights for developing efficient biocatalysts for chitin valorization.