RNF213 was originally identified as a susceptibility gene for Moyamoya disease (MMD), and its variants are also associated with other vascular occlusive disorders, including pulmonary arterial hypertension and large-artery atherosclerosis. RNF213 encodes a large protein with AAA+ ATPase and E3 ubiquitin ligase activities and has been implicated in vasculopathy as well as cellular responses to microbial infection and lipid stress. However, the precise biological functions of RNF213 and the pathogenic mechanisms underlying disease-associated mutations remain poorly understood. Recent studies have suggested lipid-induced stress may play a key role in MMD pathogenesis. Therefore, we performed temporal transcriptomic analyses to identify RNF213-regulated signaling pathways in response to palmitate, the most common saturated fatty acid. Our results demonstrate that RNF213 is required for activation of apoptosis, the unfolded protein response, and NFκB signaling, as well as for the regulation of autophagy and oxidative stress responses following palmitate exposure. These functions were dependent on both the AAA+ ATPase and RZ-finger domains of RNF213 while MMD-associated RNF213 mutations enhanced palmitate-induced signaling responses. Together, these findings suggest that gain-of-function RNF213 mutations, combined with dysregulated lipid metabolism, contribute to the pathogenesis of RNF213-associated vasculopathies.
Host-microbe interactions within the gut have been extensively reviewed in the context of host immune response. Emerging evidence, however, highlights that these inflammatory and immune outcomes are often deeply intertwined with the microbiome-derived secondary metabolites. The gut microbiota functions in concert with the host by providing an extensive repertoire of metabolic enzymes that enhance digestion and capacity to assimilate a broad spectrum of ingested food sources. This symbiotic metabolism generates a diverse array of bioactive metabolites that shape local and systemic physiology, adaptive immune responses, and neuroimmune responses. Here, we focus on microbial metabolism as a central organizing principle of host-microbiota symbiosis. Microbiota-derived metabolites, including short-chain fatty acids, secondary bile acids, tryptophan-derived indoles, sphingolipids, and gaseous byproducts, signal through downstream molecular partners like nuclear receptors, transcriptional regulators, and redox-sensitive homeostatic pathways to regulate host energy homeostasis, but also alter immune functions like gut epithelial integrity, immune tolerance, and neuroimmune crosstalk. Finally, we discuss emerging therapeutic strategies that target microbial metabolic functions-including dietary interventions, engineered probiotics, postbiotics, and receptor-directed approaches-that position microbial metabolism as a tractable axis for modulating immunometabolism homeostasis and potentially mitigating metabolic and inflammatory diseases.
The central nervous system is highly active and one of the fastest consumers of oxygen. Thus, the brain requires immediate and effective oxygenation by arterial blood supply. Singlet oxygen is a non-radical form of oxygen which can be produced in cells by non-enzymatic and enzymatic means. Generation of non-toxic doses of singlet oxygen in a primary co-culture of neurons and astrocytes by 1267 nm laser induces calcium signalling in astrocytes via the induction of lipid peroxidation, activation of phospholipase C and production of IP3. [Ca2+]i rise in astrocytes triggers fusion of vesicular compartments containing ATP which in turn leads to singlet oxygen-induced calcium waves in astrocytes. Consequently, these events increase the diameter of blood vessels in the brain. Thus, these data suggest that singlet oxygen may be involved in physiological regulation of brain blood circulation and reoxygenation.
Disrupted protein homeostasis is a shared characteristic in ageing, obesity-induced lipotoxicity and neurodegenerative diseases. The accumulation of misfolded or unfolded proteins within the cell triggers endoplasmic reticulum (ER) stress. In response, the unfolded protein response (UPR) and ER-associated degradation (ERAD) pathways are activated. A key mechanism to alleviate intracellular protein aggregation involves ubiquitination of substrates and their subsequent degradation by the proteasome. The ubiquitin-proteasome system (UPS) is indispensable for cellular protein quality control, and its dysfunction contributes to various proteopathies. However, the crosstalk between the proteasome subunit Rpt3 and the Ire1-Hac1 pathway appears to be rarely reported. In Saccharomyces cerevisiae, growth curve and spotting assay demonstrated that overexpression of Rpt3 reduced the sensitivity of ire1Δ or hac1Δ to ER stressors. The growth-promoting effect of Rpt3 is not a common feature of the BASE subunits, as overexpression of Rpt6 failed to rescue the growth inhibition. Deletion of hac1 resulted in stoichiometric imbalance among proteasomal subunits, which may be key for Rpt3-mediated rescue of hac1Δ growth, as deletion of the proteasome transcriptional factor Rpn4 impedes Rpt3 from restoring the growth of hac1Δ from ER stress. Overexpression of Rpt3 enhanced proteasome assembly and activity, reducing intracellular ubiquitin levels in hac1Δ. Moreover, Rpt3 increased the protein level of Hac1, and its alleviation of proteotoxic stress was dependent on the collaboration of ubiquitinating enzymes and chaperones. Western blot and proteasome activity assay in human cells confirmed the cross-species conservation of Rpt3 function. These results highlight a dual role for Rpt3 in proteostasis: beyond enhancing proteasomal activity, Rpt3 upregulates Hac1 protein abundance, thereby ensuring proteostasis maintenance.
Precise spatial positioning of the cytokinetic Z-ring is essential for symmetric bacterial cell division. In the ovoid bacterium Streptococcus pneumoniae, MapZ acts as a positive regulator that guides Z-ring assembly at midcell. However, many MapZ-deficient cells still maintain relatively normal Z-ring orientation, suggesting the existence of additional spatial cues. Here, we combined fluorescence microscopy with quantitative image analysis to investigate the relationship between cell elliptic ratio and Z-ring orientation across multiple mutant backgrounds. In MapZ-deficient cells, the cell elliptic ratio was negatively correlated with the Z-ring deviation angle, and genetic perturbations that reduced cell elongation increased the frequency of large-angle Z-ring misorientation. These findings suggest that cellular geometry contributes to Z-ring orientation when MapZ-dependent regulation is compromised. We propose that nucleoid organization and cellular geometry together provide spatial constraints that influence division-plane selection.
Diabetes mellitus (DM) is a metabolic disease characterised by chronic hyperglycemia. The Glucagon-Like Peptide-1 Receptor Agonists (GLP-1 RAs) used in type 2 diabetes mellitus (T2DM) have been highlighted for their potential benefits to the gut microbiota in the context of this pathology. Therefore, it is important to investigate the relationship between microbiota and GLP-1 RAs used in T2DM. This review aimed at evaluating the effects of GLP-1 agonists on gut microbiota in T2DM individuals. The protocol was previously registered in PROSPERO, and the search was conducted in PubMed, Scopus, Web of Science, and Embase databases in March 2025. Two reviewers have read the titles and abstracts and selected the included articles based on established criteria. The initial search identified 5532 publications, of which 17 studies were included: 12 preclinical and 5 clinical. Most were conducted in China between 2016 and 2025. GLP-1 agonists have been shown to modulate the gut microbiota, characterised by an increase in Bacteroidetes, Akkermansia muciniphila, and Lactobacillus. These changes are accompanied by an improvement of intestinal villi, increase in occludin expression, as well as an improvement in colonic inflammation. We identified a higher abundance of bacterial species associated with intestinal balance (eubiosis), accompanied by reduced inflammation and insulin resistance. Conversely, there was a lower abundance of bacteria related to dysbiosis, such as Firmicutes and Proteobacteria, which can be unfavourable when in excess. GLP-1 agonists influence the intestinal microbiota, improving metabolic parameters and anti-inflammatory effects. However, further clinical studies are needed to corroborate the findings of this review.
Fungal phytopathogens represent a major threat to global agriculture, causing extensive yield losses. Among them, Colletotrichum spp. are infamous for their capacity of infecting a wide variety of monocot and dicot hosts. Motivated by the recent discovery in Colletotrichum orbiculare of a redox relay between Copper Radical Alcohol Oxidases from the AA5_2 subfamily and tandem peroxidases involved in plant infection, we investigated species from the Colletotrichum species complex (C. acutatum and C. tamarilloi) where the tandem peroxidase gene is absent. Instead, putative ring-cleavage dioxygenases are found within a conserved genomic locus adjacent to AA5_2 paralogues. Therefore, we hypothesized that ring-cleavage dioxygenases could function as redox partners for AA5_2. Given the limited information available on fungal dioxygenases, we performed a biochemical characterization of both Colletotrichum ring-cleavage dioxygenases. LC-MS analyses revealed that both enzymes catalyze intradiol (1,2-) ring cleavage of catecholic substrates and preferentially oxidize methyl-substituted catechols. Structural modeling indicated an open, solvent-exposed active site resembling that of spider mite intradiol ring cleavage dioxygenases and distinct from the compact, oligomeric bacterial homologs. However, both dioxygenases showed only a limited capacity to activate AA5_2 in vitro, indicating that close genomic proximity does not necessarily imply efficient functional coupling. Together, these biochemical insights into both AA5_2 and ring-cleavage dioxygenases provide a foundation for future studies aimed at elucidating their biological roles in fungal metabolism and pathogenicity.
The glutamate delta receptors GluD1 and GluD2 are part of the ionotropic glutamate receptor (iGluR) family; however, delta receptors differ from other iGluRs as they do not bind glutamate. These receptors have important and diverse functions in the brain and are known to be involved in various neurological diseases. Here, we report the monomeric X-ray crystal structure of the ligand-binding domain of rat GluD1 (rGluD1-LBD) with D-serine and Zn2+ ions, determined at 2.8 Å resolution. By comparing the structures with Zn2+ ions (rGluD1-LBD) and Ca2+ ions (human, hGluD1-LBD), we show that species and crystallization differences do not affect intermediate domain closure and D-serine interactions with GluD1. Furthermore, examining the effect of replacing the Ca2+ ions in the hGluD1-LBD dimer with Mg2+, Zn2+ or Na+ ions did not reveal significant differences in the overall structure of the hGluD1-LBD for the different cations. We show that the cations were coordinated by the same residues, Glu527, Val530 and Asp531, and several water molecules. In the structure of hGluD1-LBD, Cl- ions are present at the dimer interface and molecular dynamics (MD) simulations showed that removal of the ions leads to opening of the dimer, highlighting the importance of Cl- ions in stabilizing the dimer. Finally, MD simulations of hGluD1-LBD and the Pro725 to Ser725 mutant, with and without D-serine in the binding site, suggest that Pro725 hinders full domain closure in GluD1, whereas an apparent synergistic effect of D-serine and mutation to Ser725 leads to a significant interlobe closure of the clamshell-like structure.
Multidomain proteins play central roles in cellular regulation, yet their intrinsic flexibility and structural instability often hinder optimization for biotechnological applications. Here, we present an integrated structure-guided and deep learning-assisted engineering strategy that combines structure modeling with Protein Message Passing Neural Network (ProteinMPNN)-based sequence design to generate an ultracompact CRISPR activator (uCRISPRa) derived from the miniature CRISPR/Cas12f. Structural and computational analyses identified flexible, nonessential regions within both Cas12f and its single-guide RNA (sgRNA), enabling rational truncation and sequence redesign while preserving DNA-targeting capability. When delivered as mRNA encapsulated in lipid nanoparticles, uCRISPRa achieved selective activation of olfr544 among more than a thousand homologous olfactory receptor genes in skeletal muscle cells, leading to enhanced mitochondrial biogenesis. These findings demonstrate that the integration of structure-based protein engineering with deep learning sequence optimization provides a powerful framework for developing compact and efficient CRISPR effectors, offering broad potential for precise gene regulation and functional studies of complex macromolecular systems.
A central question in gene regulation is the relationship between the nanoscale organization of chromatin and transcriptional activity. However, directly visualizing and quantifying this process in living cells at nanometer resolution remains challenging. Here, we integrate live-cell MINFLUX nanoscopy with the DNA probe 5-HMSiR-Hoechst and a HaloTag system to map chromatin architecture at loci colabeled with RNA polymerase II (Pol II) and heterochromatin protein 1α (HP1α)-markers of distinct transcriptional states. High-resolution snapshots, captured via spatiotemporal segmentation of single-molecule localizations, reveal that HP1α loci exhibit densely packed chromatin with high DNA localization density, indicating a stable, rigid state. In contrast, Pol II loci are predominantly open and extended and exhibit low DNA localization density, implying a highly flexible and dynamic conformation. Critically, we establish a direct positive correlation between the degree of HP1α aggregation and the level of local chromatin compaction. Our work thus provides nanoscale insight into how transcriptional activity is physically encoded in living cell chromatin.
Lecithin-cholesterol acyltransferase (LCAT), the key enzyme in reverse cholesterol transport (RCT), has been traditionally recognized for playing roles in high-density lipoprotein (HDL) maturation and systemic cholesterol homeostasis. However, its function in liver disease remains elusive. Here, we identify the role of LCAT in driving hepatic steatosis. Using a hepatocyte-specific lcat-overexpressing zebrafish model combined with transcriptomic and metabolic phenotyping, we demonstrated that lcat overexpression induces triglyceride (TG) accumulation and cytoplasmic lipid droplet deposition in the liver. Unexpectedly, transcriptomic profiling revealed an upregulation of fatty acid elongation pathway genes in lcat-overexpressed hepatocytes. Crucially, genetic knockdown or pharmacological inhibition of fatty acid elongation enzymes (hsd17b12a and Elovl1) alleviated LCAT-induced hepatic steatosis. Furthermore, overexpression of hsd17b12a and elovl1 resulted in hepatic steatosis similar to that of lcat-overexpressing larvae. These findings establish a novel role for LCAT in hepatic steatosis through regulating fatty acid metabolism, providing a mechanistic basis for targeting Lcat/fatty acid elongation axis in metabolic dysfunction-associated steatotic liver disease (MASLD).
The DEAD-box proteins of the superfamily II (SF2) RNA/DNA helicases are typically found to be ATP-dependent RNA binding proteins and RNA-dependent ATPases; however, they have poor, nonprocessive, RNA unwinding activity. Moreover, cells contain multiple variants that are associated with specific cellular processes, and they are generally not interchangeable. Thus, they can be thought of as ATP-dependent switches to control the progression and directionality of cellular metabolism and catabolism. We have characterized a DEAD-box protein (LINF08) from the trypanosomatid parasite Leishmania infantum that has homology to the spliceosome protein DDX46/Prp5. It is expressed in the nucleus of the flagellated promastigotes and the immobile axenic amastigotes. It has very high intrinsic ATPase activity that is independent of added nucleic acids, and it is activated both by the usual bound magnesium cation and by calcium, which is a cation that typically does not directly participate in catalysis. DDX46/Prp5 in other eukaryotes is involved in the early proofreading steps of spliceosome assembly to resolve structural aberrations between the U2 small nuclear RNA (snRNA) and the splice junction. Although trypanosomes typically lack introns, all of the mRNAs are transcribed as polycistronic RNAs that are processed into individual mRNAs involving a process known as SL-dependent trans splicing. Trypanosomes retain the spliceosome machinery but the snRNAs are in an abbreviated form. Thus, although LINF08 appears to be a DDX46/Prp5 homolog, it has probably evolved for the unusual splicing machinery and highly variable environment of the parasite.
Prenylated flavin mononucleotide (prFMN) is a flavin coenzyme that helps UbiD-family microbial enzymes catalyze the decarboxylation of α,β-unsaturated carboxylic acids. Since the UbiD-family decarboxylases are involved in important metabolic processes, such as the anaerobic catabolism of aromatic acids, bacterial ubiquinone biosynthesis, and the archaeal modified mevalonate pathway, prFMN is essential for many microorganisms. Biosynthesis of prFMN via the transfer of a dimethylallyl group to the N5 of reduced FMN and subsequent cyclization is catalyzed by a UbiX-family prenyltransferase called prFMN synthase (PFS). PFSs are unique because some accept dimethylallyl phosphate (DMAP) as the prenyl donor substrate instead of dimethylallyl diphosphate (DMAPP), which is a common donor substrate for many prenyltransferases. Structural and mutagenic analyses of PFS from the methanogenic archaeon Methanosarcina mazei were performed in the present study to elucidate the detailed mechanism underlying the unique donor substrate preference of PFSs. M. mazei PFS is DMAP-specific, but it can also accept DMAPP. The crystal structures of the enzyme in complex with FMN, both FMN and DMAP, or prFMN were solved, revealing the substrate-binding residues. Point mutations at a non-conserved residue, Thr163, near the substrate-binding site changed the donor substrate specificity, primarily affecting the catalytic rate rather than substrate recognition. The T163F mutant significantly decreased its activity toward DMAPP, becoming more specific to DMAP, whereas the T163Q mutant was completely inactive when DMAPP was used for the reaction. This study provides a deeper understanding of how PFS recognizes its substrates and synthesizes prFMN, emphasizing the importance of DMAP, an overlooked metabolite.
Frataxin is a mitochondrial iron-binding protein whose deficiency causes Friedreich's ataxia, yet the dynamic mechanisms by which this protein communicates iron-binding events to distal regions remain poorly understood. Traditional correlation-based analyses identify coupled residue motions but cannot resolve the directionality of information flow, leaving critical mechanistic gaps. Here we employ transfer entropy analysis of molecular dynamics simulations (1.5 μs) to map the complete allosteric network in human frataxin, validated through orthogonal experimental approaches. We identify LEU47 (LEU136 in UniProt Q16595 numbering) and LEU51 (LEU140) as primary signal sources with net transfer entropy values of 0.415 and 0.249, respectively, connecting the hydrophobic core to the iron-binding acidic ridge. NMR relaxation at 600 and 800 MHz reveals elevated R2/R1 ratios (9.90-10.00) and significant exchange contributions (Rex = 3-5 s-1) specifically at these primary signal source residues, indicating μs-ms dynamics. Hydrogen-deuterium exchange mass spectrometry demonstrates that hub residues possess intermediate protection factors (ln(PF) = 5.97-6.07) optimal for conformational signaling, while iron binding induces bidirectional protection changes propagating through the identified pathway. Systematic mutagenesis confirms that disruption of hub residues reduces iron-binding affinity 1.9-4.2-fold and decreases thermal stability by 4.3-11.2 °C, despite occupying buried-core positions distant from the iron-coordinating acidic-ridge residues (LEU136/LEU140 Cα to ASP122, ASP124, and GLU189 = 6.7 to 11.8 Å in PDB 1EKG). The strong prediction-experiment correlation establishes transfer entropy as a reliable predictor of functionally important allosteric residues and provides a methodological framework applicable to other proteins of biomedical significance.
The bacterium Rubellimicrobium mesophilum possesses a blue light-using flavin (BLUF)-coupled endonuclease III (BLUF-EndoIII) with potential endonuclease activity. Interestingly, the crucial amino acid residues (tyrosine, histidine, and tryptophan) responsible for BLUF photocycle and photodynamics are replaced by phenylalanine (Y5F), asparagine (H27N), and alanine (W87A) residues, respectively. In the present study, we investigated the impact of this evolutionary plasticity on BLUF photodynamics and the associated endonuclease activity. The results obtained showed that the residue exchanges within the BLUF domain influenced its functional aspects, including flavin binding, domain stability, recovery kinetics, and spectral characteristics. The evolutionary plasticity-induced changes in the flavin-binding pocket of the BLUF domain elevated the light-gated endonuclease activity associated with the EndoIII domain. Molecular docking analysis and spectroscopic studies confirmed the substrate-binding ability of BLUF-EndoIII. Elevated endonuclease activity suggested that the amino acid residues, previously deemed crucial for the BLUF photocycle, are indeed dispensable, and there might exist another pathway for BLUF domain activation and regulation of the associated endonuclease domain. Considering the role of endonucleases in bacterial defense, it is crucial to understand the BLUF photodynamics and mechanism of signal transfer to the downstream endonuclease domain. This understanding elucidates the functioning of the naturally occurring light-gated endonuclease BLUF-EndoIII in the bacterium Rubellimicrobium mesophilum.
With the aim of identifying vulnerabilities of subpopulation-specific metabolic networks, we used single-cell transcriptomics to build mRNA-based metabolic maps for 5512 cells from four proliferating cell populations (three liver cancer-related and a control). Paradoxically, flux balance analysis (FBA) predicted no growth for any cell. Further analysis confirmed that this conclusion was not due to shared metabolites or insufficient mRNA sequencing depth. Instead, using our enzyme-based Vmax-constrained model, FBA showed that each cell population should support biomass production for division, suggesting an enzyme-stability based explanation of growth of individual cells, notwithstanding their apparent mRNA insufficiency. Comparing liver cancer cells and noncancerous liver cells via cell-population-average mRNA data, we identified cholesterol synthesis (SQLEr) and arginine synthesis as potential drug targets. Therefore, we suggest that identifying subclusters and their vulnerabilities reveals new drug targets for subpopulations, which should enable the reduction of drug resistance.
Immune checkpoint inhibitors (ICIs), which restore antitumor immunity mediated by T cells, have caused a paradigm shift in cancer therapy. However, only a subset of patients achieve durable clinical responses, highlighting the need to better understand the mechanisms that determine antitumor immunity. Cancer-specific neoantigens are considered key determinants of effective immune responses because they are recognized as non-self by T cells and can induce strong immune responses. Accordingly, neoantigens are considered promising targets for cancer vaccines and adoptive cell therapies. Neoantigens have conventionally been considered to arise from somatic mutations and have been identified using whole-exome sequencing (WES). However, the number of neoantigens identified with WES remains limited. In contrast, advances in technology have revealed that peptides derived from various sources, including noncoding regions, are immunogenic and function as neoantigens. In particular, human leukocyte antigen (HLA) ligandome analyses, which enabled the direct identification of peptides presented on HLA molecules in combination with various sequencing technologies, have extended the landscape of neoantigen sources. However, in many cases, the immunogenicity of the identified neoantigen candidates has not been experimentally evaluated. Recently, several experimental platforms have been developed to validate neoantigen immunogenicity, which can be complemented by single-cell RNA/T-cell receptor sequencing to identify tumor-reactive T cell clones. In this review, we summarize current neoantigen identification strategies and discuss the challenges and future perspectives in neoantigen research.
Glutathione peroxidases (Gpx) are well-characterized antioxidative enzymes in eukaryotes; however, their bacterial counterparts remain poorly explored. Nevertheless, bacterial Gpx homologs play pivotal role in pathogenesis. Staphylococcus aureus encodes two conserved Gpx homologs but lacks glutathione (GSH) and the glutathione reductase enzyme required for canonical Gpx function. Herein, we aimed to elucidate the structure and function of one of the S. aureus Gpx homologs (SaGpx, EC:1.11.1.9, Uniprot Id: Q2FYZ0). We solved the high-resolution (1.55 Å) crystal structure of the SaGpx C36S mutant, which demonstrates SaGpx adopts a canonical Gpx fold with a conserved catalytic tetrad composed of C36, Q70, W124 and N125. The crystal structure of SaGpx C36S, demonstrates a profound structural similarity with the mammalian Gpx4. The structure of SaGpx resembles a fully folded, reduced conformational state of the enzyme's active site with an intensive hydrogen-bonding network among the catalytic tetrad, required for the generation of cysteine thiolate nucleophile and for substrate binding. This analysis of the wild-type SaGpx and its active site amino acid mutants unravels the mechanistic details of its plausible catalytic mechanism. Instead of GSH, SaGpx prefers to utilize the reduced Staphylococcal thioredoxin1 as its cognate electron donor. The catalytic mechanism involved the formation of a cysteine sulfenic acid intermediate, followed by the formation of an intramolecular disulfide bond (between C36 and C82), which is subsequently resolved by thioredoxin1. This work provides the first structure-based biochemical characterization of a bacterial glutathione peroxidase homolog, establishing the novel structural insights of SaGpx as a noncanonical thioredoxin-dependent glutathione peroxidase.
The effects of extracellular fluid viscosity on cell behavior are increasingly appreciated. Smith et al. observed that cells respond differently to two polymers that generate identical fluid viscosity, suggesting that molecular origins of viscosity shape cellular behavior. One possible explanation is that differences in interpolymer interaction arising from a polymer's charge distribution produce Newtonian vs Non-Newtonian rheology, thereby changing the interactions with cell membrane and contributing to distinct cellular responses.
The CldA enzyme is an unprecedented functional intermediate exhibiting the dual hydrolytic specificity of starch hydrolases and the intramolecular transglycosylation capacity of cyclomaltodextrin glucanotransferases (CGTases) from subfamily 2 of family 13 of glycoside hydrolases (GH13_2). Here, the crystallographic structure of CldA was determined at 1.66 Å resolution. Structural and kinetic studies revealed that the thermophilic CldA has a three-domain ABC architecture similar to that of starch hydrolases from GH13_1, and it contains three calcium-dependent folding centers (Ca+2 1-3) essential for thermostability. However, it simultaneously features nine expanded subsites (-7 to +2) defining the active site cleft of the canonical five-domain ABCDECBM20 CGTases from GH13_2. Structural comparisons revealed three evolutionary adaptations in CldA: (a) the absence of the substrate-guiding DECBM20 domains; (b) an unusual hydrophobic pair, Trp204/Met281, whose hydrophobicity was critical for stabilizing the cyclodextrin (CD) ring at the acceptor subsite +2; and (c) an unexpected hydrogen bond of 2.60 Å between Ser200 at subsite -6 and the key central aromatic residue, Phe216, involved in starch circularization for CD formation. Characterization of two mutants, CldAM281F and CldAS200G, and a five-domain chimera, CldA-DECBM20, provided insights into the boundary between starch hydrolases and CGTases. CldA provides the first experimental structural evidence for a native GH13_2 enzyme where hydrolytic and cyclization activities coexist at a presculpted CGTase active site. Overall, structural and functional analysis of CldA showed that it diverges from canonical GH13_2 CGTases by lacking C-terminal DE domains, shifting its intramolecular transglycosylation specificity toward hydrolysis through an intriguing starch concentration-dependent product-length mechanism.
