Natural history study of hepatic glycogen storage disease type IV and comparison to Gbe1ys/ys model.

BackgroundGlycogen storage disease type IV (GSD IV) is an ultrarare autosomal recessive disorder that causes deficiency of functional glycogen branching enzyme and formation of abnormally structured glycogen termed polyglucosan. GSD IV has traditionally been categorized based on primary hepatic or neuromuscular involvement, with hepatic GSD IV subclassified as discrete subtypes: classic (progressive) and nonprogressive.MethodsTo better understand the progression of liver disease in GSD IV, we present clinical and histopathology data from 23 patients from around the world and characterized the liver involvement in the Gbe1ys/ys knockin mouse model.ResultsWe propose an alternative to the established subtype-based terminology for characterizing liver disease in GSD IV and recognize 3 tiers of disease severity: (i) "severe progressive" liver disease, (ii) "intermediate progressive" liver disease, and (iii) "attenuated" liver disease. Analysis of liver pathology revealed that risk for liver failure cannot be predicted from liver biopsy findings alone in individuals affected by GSD IV. Moreover, analysis of postmortem liver pathology from an individual who died over 40 years after being diagnosed with nonprogressive hepatic GSD IV in childhood verified that liver fibrosis did not regress. Last, characterization of the liver involvement in a mouse model known to recapitulate the adult-onset neurodegenerative form of GSD IV (Gbe1ys/ys mouse model) demonstrated hepatic disease.ConclusionOur findings challenge the established subtype-based view of GSD IV and suggest that liver disease severity among patients with GSD IV represents a disease continuum.Trial registrationClinicalTrials.gov NCT02683512FundingNone.

Synergism of dual AAV gene therapy and rapamycin rescues GSDIII phenotype in muscle and liver.

Glycogen storage disease type III (GSDIII) is a rare metabolic disorder due to glycogen debranching enzyme (GDE) deficiency. Reduced GDE activity leads to pathological glycogen accumulation responsible for impaired hepatic metabolism and muscle weakness. To date, there is no curative treatment for GSDIII. We previously reported that 2 distinct dual AAV vectors encoding for GDE were needed to correct liver and muscle in a GSDIII mouse model. Here, we evaluated the efficacy of rapamycin in combination with AAV gene therapy. Simultaneous treatment with rapamycin and a potentially novel dual AAV vector expressing GDE in the liver and muscle resulted in a synergic effect demonstrated at biochemical and functional levels. Transcriptomic analysis confirmed synergy and suggested a putative mechanism based on the correction of lysosomal impairment. In GSDIII mice livers, dual AAV gene therapy combined with rapamycin reduced the effect of the immune response to AAV observed in this disease model. These data provide proof of concept of an approach exploiting the combination of gene therapy and rapamycin to improve efficacy and safety and to support clinical translation.

Replacement of Loops at the Entrance of the Active Pocket of Streptococcus thermophilus 4,6-α-Glucanotransferase Changes Its Catalytic Activity and Product Specificity.

To explore the roles of loops around active pocket in the reuteran type 4,6-α-glucanotransferase (StGtfB) from S. thermophilus, they were individually or simultaneously replaced with those of an isomalto/maltopolysaccharides type 4,6-α-glucanotransferase from L. reuteri. StGtfB with the replaced loops A1, A2 (A1A2) and A1, A2, B (A1A2B), respectively, showed 1.41- and 0.83-fold activities of StGtfB. Two mutants reduced crystallinity and increased starch disorder at 2, 4, and 8 U/g more than StGtfB and increased DP ≤ 5 short branches of starch by 38.01% at 2 U/g, much more than StGtfB by 4.24%. A1A2B modified starches had the lowest retrogradation over 14 days. A1A2 modified starches had the highest percentage of slowly digestible fractions, ranging from 40.32% to 43.34%. StGtfB and its mutants bind substrates by hydrogen bonding and van der Waals forces at their nonidentical amino acid residues, suggesting that loop replacement leads to a different conformation and changes activity and product structure.

Tailor-Made α-Glucans by Engineering the Processivity of α-Glucanotransferases via Tunnel-Cleft Active Center Interconversions.

The function of polysaccharides is intimately associated with their size, which is largely determined by the processivity of transferases responsible for their synthesis. A tunnel active center architecture has been recognized as a key factor that governs processivity of several glycoside hydrolases (GHs), e.g., cellulases and chitinases. Similar tunnel architecture is also observed in the Limosilactobacillus reuteri 121 GtfB (Lr121 GtfB) α-glucanotransferase from the GH70 family. The molecular element underpinning processivity of these transglucosylases remains underexplored. Here, we report the synthesis of the smallest (α1 → 4)-α-glucan interspersed with linear and branched (α1 → 6) linkages by a novel 4,6-α-glucanotransferase from L. reuteri N1 (LrN1 GtfB) with an open-clefted active center instead of the tunnel structure. Notably, the loop swapping engineering of LrN1 GtfB and Lr121 GtfB based on their crystal structures clarified the impact of the loop-mediated tunnel/cleft structure at the donor subsites -2 to -3 on processivity of these α-glucanotransferases, enabling the tailoring of both product sizes and substrate preferences. This study provides unprecedented insights into the processivity determinants and evolutionary diversification of GH70 α-glucanotransferases and offers a simple route for engineering starch-converting α-glucanotransferases to generate diverse α-glucans for different biotechnological applications.

Insights into the Structure-Function Relationship of GH70 GtfB α-Glucanotransferases from the Crystal Structure and Molecular Dynamic Simulation of a Newly Characterized Limosilactobacillus reuteri N1 GtfB Enzyme.

α-Glucanotransferases of the CAZy family GH70 convert starch-derived donors to industrially important α-glucans. Here, we describe characteristics of a novel GtfB-type 4,6-α-glucanotransferase of high enzyme activity (60.8 U mg-1) from Limosilactobacillus reuteri N1 (LrN1 GtfB), which produces surprisingly large quantities of soluble protein in heterologous expression (173 mg pure protein per L of culture) and synthesizes the reuteran-like α-glucan with (α1 → 6) linkages in linear chains and branch points. Protein structural analysis of LrN1 GtfB revealed the potential crucial residues at subsites -2∼+2, particularly H265, Y214, and R302, in the active center as well as previously unidentified surface binding sites. Furthermore, molecular dynamic simulations have provided unprecedented insights into linkage specificity hallmarks of the enzyme. Therefore, LrN1 GtfB represents a potent enzymatic tool for starch conversion, and this study promotes our knowledge on the structure-function relationship of GH70 GtfB α-glucanotransferases, which might facilitate the production of tailored α-glucans by enzyme engineering in future.

N1019D Mutant of Limosilactobacillus reuteri 121 4,6-α-Glucanotransferase GtfB Significantly Improved Catalytic Activity.

Limosilactobacillus reuteri 121 4,6-α-glucanotransferase GtfB (Lr 121 GtfB), belonging to glycoside hydrolase family 70 (GH70), synthesizes linear isomalto/malto polysaccharides having (α1→6) linkages attached to the nonreducing ends of (α1→4) linked maltose oligosaccharide segments using starch or maltodextrin as a substrate. Since Lr 121 GtfB has low catalytic activity and efficiency, it leads to substrate regeneration and reduced substrate utilization. In this study, we superimposed the crystal structure of Lr 121 GtfB-ΔNΔV with that of L. reuteri NCC 2613 GtfB-ΔNΔV (Lr 2613 GtfB-ΔNΔV) to identify the acceptor binding subsites +1 to +3 and constructed five single-residue mutants and a random mutagenesis of N1019. Compared with the wild-type, N1019D Lr 121 GtfB-ΔN did not alter the product specificity, increased the catalytic activity and efficiency by 420 and 590%, respectively, and maintained >80% relative activity in the pH 3.5-6.5 interval. The findings will contribute to the industrial application of Lr 121 GtfB and provide new solutions for starch synthesis of higher value derivatives.

Enhancement of ß-Cyclodextrin Production Using a Glycogen Debranching Enzyme from Saccharolobus solfataricus STB09.

Efficient production of cyclodextrins (CDs) has always been challenging. CDs are primarily produced from starch via cyclodextrin glycosyltransferase (CGTase), which acts on α-1,4 glucosidic bonds; however, α-1,6 glucosidic bonds in starch suppress the enzymatic production of CDs. In this study, a glycogen debranching enzyme from Saccharolobus solfataricus STB09 (SsGDE) was utilized to promote the production of ß-CD by hydrolyzing α-1,6 glucosidic bonds. The addition of SsGDE (750 U/g of starch) at the liquefaction stage remarkably improved the ß-CD yield, with a 43.9% increase. Further mechanism exploration revealed that SsGDE addition could hydrolyze specific branches with less generation of byproducts, thereby promoting CD production. The chain segments of a degree of polymerization ≥13 produced by SsGDE debranching could also be utilized by ß-CGTase to convert into CDs. Overall, these findings proposed a new approach of combining SsGDE with ß-CGTase to enhance the CD yield.

Exploring a GtfB-Type 4,6-α-Glucanotransferase to Synthesize the (α1 → 6) Linkages in Linear Chain and Branching Points from Amylose and Enhance the Functional Property of Granular Corn Starches.

Starch-converting α-glucanotransferases of glycoside hydrolase family 70 (GH70) are promising enzymatic tools for the production of diverse α-glucans with (potential) commercial applications in food and health and as biomaterials. In this study, a novel GtfB enzyme from Weissella confusa MBF8-1 was screened in the National Center for Biotechnology Information (NCBI) nonredundant protein database. The enzyme (named WcMBF8-1 GtfB) displayed high conservation in motifs I-IV with other GtfB enzymes but possessed unique variations in several substrate-binding residues. Structural characterizations of its α-glucan products revealed that WcMBF8-1 GtfB exhibited an atypical 4,6-α-glucanotransferase activity and was capable of catalyzing, by cleaving off (α1 → 4)-linkages in starch-like substrates and the synthesis of linear (α1 → 6) linkages and (α1 → 4,6) branching points. The product specificity enlarges the diversity of α-glucans and facilitates recognition of the determinants of the linkage specificity in GtfB enzymes. Furthermore, the contents of slowly digestible starch and resistant starch of granular corn starches, modified by WcMBF8-1 GtfB, increased by 6.7%, which suggested the potential value for the utilization of WcMBF8-1 GtfB to prepare "clean-label" starch ingredients with improved functional attributes.

A functional mini-GDE transgene corrects impairment in models of glycogen storage disease type III.

Glycogen storage disease type III (GSDIII) is a rare inborn error of metabolism affecting liver, skeletal muscle, and heart due to mutations of the AGL gene encoding for the glycogen debranching enzyme (GDE). No curative treatment exists for GSDIII. The 4.6 kb GDE cDNA represents the major technical challenge toward the development of a single recombinant adeno-associated virus-derived (rAAV-derived) vector gene therapy strategy. Using information on GDE structure and molecular modeling, we generated multiple truncated GDEs. Among them, an N-terminal-truncated mutant, ΔNter2-GDE, had a similar efficacy in vivo compared with the full-size enzyme. A rAAV vector expressing ΔNter2-GDE allowed significant glycogen reduction in heart and muscle of Agl-/- mice 3 months after i.v. injection, as well as normalization of histology features and restoration of muscle strength. Similarly, glycogen accumulation and histological features were corrected in a recently generated Agl-/- rat model. Finally, transduction with rAAV vectors encoding ΔNter2-GDE corrected glycogen accumulation in an in vitro human skeletal muscle cellular model of GSDIII. In conclusion, our results demonstrated the ability of a single rAAV vector expressing a functional mini-GDE transgene to correct the muscle and heart phenotype in multiple models of GSDIII, supporting its clinical translation to patients with GSDIII.

Bioinformatics and functional selection of GH77 4-α-glucanotransferases for potato starch modification.

4-α-glucanotransferases (4αGTs, EC 2.4.1.25) from glycoside hydrolase family 77 (GH77) catalyze chain elongation of starch amylopectin chains and can be utilized to structurally modify starch to tailor its gelation properties. The potential relationship between the structural design of 4αGTs and functional starch modification is unknown. Here, family GH77 was mined in silico for enzyme candidates based on sub-grouping guided by Conserved Unique Peptide Patterns (CUPP) bioinformatics categorization. From + 12,000 protein sequences a representative set of 27 4αGTs, representing four different domain architectures, different bacterial origins and diverse CUPP groups, was selected for heterologous expression and further study. Most of the enzymes catalyzed starch modification, but their efficacies varied substantially. Five of the 4αGTs were characterized in detail, and their action was compared to that of the industrial benchmark enzyme, Tt4αGT (CUPP 77_1.2), from Thermus thermophilus. Reaction optima of the five 4αGTs ranged from ∼40-60 °C and pH 7.3-9.0. Several were stable for a minimum 4 h at 70 °C. Domain architecture type A proteins, consisting only of a catalytic domain, had high thermal stability and high starch modification ability. All five novel 4αGTs (and Tt4αGT) induced enhanced gelling of potato starch. One, At4αGT from Azospirillum thermophilum (CUPP 77_2.4), displayed distinct starch modifying abilities, whereas T24αGT from Thermus sp. 2.9 (CUPP 77_1.2) modified the starch similarly to Tt4αGT, but slightly more effectively. T24αGT and At4αGT are thus interesting candidates for industrial starch modification. A model is proposed to explain the link between the 4αGT induced molecular modifications and macroscopic starch gelation.

Generation of three induced pluripotent stem cell lines from patients with glycogen storage disease type III.

Glycogen storage disease type III (GSDIII) is an autosomal recessive disorder characterized by a deficiency of glycogen debranching enzyme (GDE) leading to cytosolic glycogen accumulation and inducing liver and muscle pathology. Skin fibroblasts from three GSDIII patients were reprogrammed into induced pluripotent stem cells (iPSCs) using non-integrated Sendai virus. All of the three lines exhibited normal morphology, expression of pluripotent markers, stable karyotype, potential of trilineage differentiation and absence of GDE expression, making them valuable tools for modeling GSDIII disease in vitro, studying pathological mechanisms and investigating potential treatments.

Distribution of Aromatic Amino Acid Residues in Substrate-Binding Regions Modulates Substrate Specificity of Microbial Debranching Enzymes.

Debranching enzymes (DBEs) directly hydrolyze α-1,6-glucosidic linkages in glycogen, starch, and related polysaccharides, making them important in the starch processing industry. However, the ambiguous substrate specificity usually restricts synergistic catalysis with other amylases for improving starch utilization. Herein, a glycogen-debranching enzyme from Saccharolobus solfataricus (SsGDE) and two isoamylases from Pseudomonas amyloderamosa (PaISO) and Chlamydomonas reinhardtii (CrISO) were used to investigate the molecular mechanism of substrate specificity. Along with the structure-based computational analysis, the aromatic residues in the substrate-binding region of DBEs played an important role in binding substrates. The aromatic residues in SsGDE appeared clustered, contributing to a small substrate-binding region. In contrast, the aromatic residues in isoamylase were distributed dispersedly, forming a large active site. The distinct characteristics of substrate-binding regions in SsGDE and isoamylase might explain their substrate preferences for maltodextrin and amylopectin, respectively. By modulating the substrate-binding region of SsGDE, variants Y323F and V375F were obtained with significantly enhanced activities, and the activities of Y323F and V375F increased by 30 and 60% for amylopectin, and 20 and 23% for DE4 maltodextrin, respectively. This study revealed the molecular mechanisms underlying the substrate specificity for SsGDE and isoamylases, providing a route for engineering enzymes to achieve higher catalytic performance.

The Role of a Loop in the Non-catalytic Domain B on the Hydrolysis/Transglycosylation Specificity of the 4-α-Glucanotransferase from Thermotoga maritima.

The mechanism by which glycoside hydrolases control the reaction specificity through hydrolysis or transglycosylation is a key element embedded in their chemical structures. The determinants of reaction specificity seem to be complex. We looked for structural differences in domain B between the 4-α-glucanotransferase from Thermotoga maritima (TmGTase) and the α-amylase from Thermotoga petrophila (TpAmylase) and found a longer loop in the former that extends towards the active site carrying a W residue at its tip. Based on these differences we constructed the variants W131G and the partial deletion of the loop at residues 120-124/128-131, which showed a 11.6 and 11.4-fold increased hydrolysis/transglycosylation (H/T) ratio relative to WT protein, respectively. These variants had a reduction in the maximum velocity of the transglycosylation reaction, while their affinity for maltose as the acceptor was not substantially affected. Molecular dynamics simulations allow us to rationalize the increase in H/T ratio in terms of the flexibility near the active site and the conformations of the catalytic acid residues and their associated pKas.

Structural characterization of potato starch modified by a 4,6-α-glucanotransferase B from Lactobacillus reuteri E81.

The recent reports have revealed that increase in amount of α-1,6 linkages by modification of potato starch with enzyme (glycosyltransferases) treatment gains slowly digestible properties to the starch; however, the formation of new α-1,6-glycosidic linkages diminish the thermal resistance of the starch granules. In this study, a putative GtfB-E81, (a 4,6-α-glucanotransferase-4,6-αGT) from L. reuteri E81 was firstly used to produce a short length of α-1,6 linkages. NMR results revealed that external short chains mostly comprised of 1-6 glucosyl units were newly produced in potato starch, and the α-1,6 linkage ratio was significantly increased from 2.9 % to 36.8 %, suggesting that this novel GtfB-E81 might have potentially an efficient transferase activity. In our study, native and GtfB-E81 modified starches showed fundamental similarities with respect to their molecular properties and treatment of native potato starch with GtfB-E81 did not remarkably change thermal stability of the potato starch, which seems to be very prominent for the food industry given the significantly decreased thermal stability results obtained for the enzyme modified starches reported in the literature. Therefore, the results of this study should open up emerging perspectives for regulating slowly digestible characteristics of potato starch in future studies without a significant change in the molecular, thermal, and crystallographic properties.

Molecular mechanisms of glycogen particle assembly in Escherichia coli.

It has been reported that glycogen in Escherichia coli has two structural states, that is, fragility and stability, which alters dynamically. However, molecular mechanisms behind the structural alterations are not fully understood. In this study, we focused on the potential roles of two important glycogen degradation enzymes, glycogen phosphorylase (glgP) and glycogen debranching enzyme (glgX), in glycogen structural alterations. The fine molecular structure of glycogen particles in Escherichia coli and three mutants (ΔglgP, ΔglgX and ΔglgP/ΔglgX) were examined, which showed that glycogen in E. coli ΔglgP and E. coli ΔglgP/ΔglgX were consistently fragile while being consistently stable in E. coli ΔglgX, indicating the dominant role of GP in glycogen structural stability control. In sum, our study concludes that glycogen phosphorylase is essential in glycogen structural stability, leading to molecular insights into structural assembly of glycogen particles in E. coli.

Multiple approaches of loop region modification for thermostability improvement of 4,6-α-glucanotransferase from Limosilactobacillus fermentum NCC 3057.

4,6-α-glucanotransferase (4,6-α-GT), as a member of the glycoside hydrolase 70 (GH70) family, converts starch/maltooligosaccharides into α,1-6 bond-containing α-glucan and possesses potential applications in food, medical and related industries but does not satisfy the high-temperature requirement due to its poor thermostability. In this study, a 4,6-α-GT (ΔGtfB) from Limosilactobacillus fermentum NCC 3057 was used as a model enzyme to improve its thermostability. The loops of ΔGtfB as the target region were optimized using directed evolution, sequence alignment, and computer-aided design. A total of 11 positive mutants were obtained and iteratively combined to obtain a combined mutant CM9, with high resistance to temperature (50 °C). The activity of mutant CM9 was 2.08-fold the activity of the wild type, accompanied by a 5 °C higher optimal temperature, a 5.76 °C higher melting point (Tm, 59.46 °C), and an 11.95-fold longer half-life time (t1/2). The results showed that most of the polar residues in the loop region of ΔGtfB are mutated into rigid proline residues. Molecular dynamics simulation demonstrated that the root mean square fluctuation of CM9 significantly decreased by "Breathing" movement reduction of the loop region. This study provides a new strategy for improving the thermostability of 4,6-α-GT through rational loop region modification.

Impact of Starch Binding Domain Fusion on Activities and Starch Product Structure of 4-α-Glucanotransferase.

A broad range of enzymes are used to modify starch for various applications. Here, a thermophilic 4-α-glucanotransferase from Thermoproteus uzoniensis (TuαGT) is engineered by N-terminal fusion of the starch binding domains (SBDs) of carbohydrate binding module family 20 (CBM20) to enhance its affinity for granular starch. The SBDs are N-terminal tandem domains (SBDSt1 and SBDSt2) from Solanum tuberosum disproportionating enzyme 2 (StDPE2) and the C-terminal domain (SBDGA) of glucoamylase from Aspergillus niger (AnGA). In silico analysis of CBM20s revealed that SBDGA and copies one and two of GH77 DPE2s belong to well separated clusters in the evolutionary tree; the second copies being more closely related to non-CAZyme CBM20s. The activity of SBD-TuαGT fusions increased 1.2-2.4-fold on amylose and decreased 3-9 fold on maltotriose compared with TuαGT. The fusions showed similar disproportionation activity on gelatinised normal maize starch (NMS). Notably, hydrolytic activity was 1.3-1.7-fold elevated for the fusions leading to a reduced molecule weight and higher α-1,6/α-1,4-linkage ratio of the modified starch. Notably, SBDGA-TuαGT and-SBDSt2-TuαGT showed Kd of 0.7 and 1.5 mg/mL for waxy maize starch (WMS) granules, whereas TuαGT and SBDSt1-TuαGT had 3-5-fold lower affinity. SBDSt2 contributed more than SBDSt1 to activity, substrate binding, and the stability of TuαGT fusions.

Extracellular recombinant production of 4,6 and 4,3 α-glucanotransferases in Lactococcus lactis.

4,6 α-Glucanotransferase (4,6-α-GTase) and 4,3 α-glucanotransferases (4,3-α-GTase) produced by Lactic Acid Bacteria (LAB) in the GH70 enzyme family have become important due to their catalytic effect on starch and maltodextrins. Their high level of production is necessary for their application at industrial scale. In this respect, both enzymes were expressed extracellularly using Lactococcus lactis as GRAS host. 4,6-α-GTase and 4,3-α-GTase genes from Limosilactobacillus reuteri E81 and Limosilactobacillus fermentum PFC282 respectively were transformed into the plasmid pLEB124 vector having the signal peptide usp45 under the P45 continuous promoter and successfully expressed in Lactococcus lactis MG1363. Western blot screening showed that the relevant enzymes were able to be successfully secreted extracellularly. The Vmax and Km of 4,6-α-GTase were 2.58 µmol min-1 and 0054 mg min-1 whereas 3369 µmol min-1 and 0032 mg min-1 for 4,3-α-GTase respectively. NMR analysis demonstrated the formation of new bonds within the corresponding enzymes. Also, both enzymes were active on maltose, maltoheptaose, maltohexaose and starch and produced malto-oligosaccarides observed by TLC analysis. In conclusion, this study demonstrated first time the extracellular production of 4,6-α-GTase and 4,3-α-GTase with GRAS status that can be useful for starch retrogradation delay and glycaemic index reduction.

Abundant copathologies of polyglucosan bodies, frontotemporal lobar degeneration with TDP-43 inclusions and ageing-related tau astrogliopathy in a family with a GBE1 mutation.

AIMS: Adult polyglucosan body disease (APBD) is a progressive neurogenetic disorder caused by 1,4-alpha-glucan branching enzyme 1 (GBE1) mutation with an accumulation of polyglucosan bodies (PBs) in the central and peripheral nervous systems as a pathological hallmark. Here, we report two siblings in a family with a GBE1 mutation with prominent frontotemporal lobar degeneration with TAR DNA-binding protein 43 (FTLD-TDP) and ageing-related tau astrogliopathy (ARTAG) copathologies with PBs in the central nervous system. METHODS: Whole-genome sequencing (WGS) followed by Sanger sequencing (SS) was performed on three affected and two unaffected siblings in a pedigree diagnosed with familial frontotemporal dementia. Out of the affected siblings, autopsies were conducted on two cases, and brain samples were used for biochemical and histological analyses. Brain sections were stained with haematoxylin and eosin and immunostained with antibodies against ubiquitin, tau, amyloid ß, α-synuclein, TDP-43 and fused in sarcoma (FUS). RESULTS: A novel single nucleotide deletion in GBE1, c.1280delG, was identified, which is predicted to result in a reading frameshift, p.Gly427Glufs*9. This variant segregated with disease in the family, is absent from population databases and is predicted to cause loss of function, a known genetic mechanism for APBD. The affected siblings showed a greater than 50% decrease in GBE protein levels. Immunohistochemical analysis revealed widespread FTLD-TDP (type A) and ARTAG pathologies as well as PBs in the brains of two affected siblings for whom an autopsy was performed. CONCLUSIONS: This is the first report of a family with several individuals with a FTD clinical phenotype and underlying copathologies of APBD, FTLD-TDP and ARTAG with a segregating GBE1 loss-of-function mutation in affected siblings. The finding of copathologies of APBD and FTLD-TDP suggests these processes may share a disease mechanism resulting from this GBE1 mutation.

Modulation of Flexible Loops in Catalytic Cavities Reveals the Thermal Activation Mechanism of a Glycogen-Debranching Enzyme.

Some thermophilic enzymes not only exhibit high thermostability at high temperatures but also have an activation effect by thermal incubation. However, the correlations between temperature-induced structural modulation and thermal activation are still unclear. In this study, we selected a thermophilic glycogen-debranching enzyme from Saccharolobus solfataricus STB09 (SsGDE), which was a promising starch-debranching enzyme with a thermal activation property at temperatures ranging from 50 to 70 °C, to explore the thermal activation mechanism. Molecular dynamics simulations were performed for SsGDE at 30, 50, or 70 °C to reveal the temperature dependence of structure modulation and catalytic function. The results revealed that four loops (loop1 313-337, loop2 399-418, loop3 481-513, and loop4 540-574) in SsGDE were reshaped, which made the catalytic cavity more open. The internal residues, including the catalytic triad Asp3631, Glu399, and Asp471, could be exposed, due to the structural modulation, to exert catalytic functions. We proposed that the thermal activation effect of SsGDE was closely associated with the temperature-induced modulation of the catalytic cavity, which paved the way for further engineering enzymes to achieve higher catalytic performance and stability.