TIM Barrel Research Articles

Crystal Structure of Allantoinase from Escherichia coli BL21: A Molecular Insight into a Role of the Active Site Loops in Catalysis

Allantoinase (ALLase; EC 3.5.2.5) possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carbamylated lysine. ALLase acts as a key enzyme for the biogenesis and degradation of ureides by catalyzing the conversion of allantoin into allantoate. Biochemically, ALLase belongs to the cyclic amidohydrolase family, which also includes dihydropyrimidinase, dihydroorotase, hydantoinase (HYDase), and imidase. Previously, the crystal structure of ALLase from Escherichia coli K-12 (EcALLase-K12) was reported; however, the two active site loops crucial for substrate binding were not determined. This situation would limit further docking and protein engineering experiments. Here, we solved the crystal structure of E. coli BL21 ALLase (EcALLase-BL21) at a resolution of 2.07 Å (PDB ID 8HFD) to obtain more information for structural analyses. The structure has a classic TIM barrel fold. As compared with the previous work, the two missed active site loops in EcALLase-K12 were clearly determined in our structure of EcALLase-BL21. EcALLase-BL21 shared active site similarity with HYDase, an important biocatalyst for industrial production of semisynthetic penicillin and cephalosporins. Based on this structural comparison, we discussed the functional role of the two active site loops in EcALLase-BL21 to better understand the substrate/inhibitor binding mechanism for further biotechnological and pharmaceutical applications.

Structural basis for the substrate specificity switching of lysoplasmalogen-specific phospholipase D from Thermocrispum sp. RD004668.

Lysoplasmalogen-specific phospholipase D (LyPls-PLD) hydrolyzes choline lysoplasmalogen to choline and 1-(1-alkenyl)-sn-glycero-3-phosphate. Mutation of F211 to leucine altered its substrate specificity from lysoplasmalogen to 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (lysoPAF). Enzymes specific to lysoPAF have good potential for clinical application, and understanding the mechanism of their activity is important. The crystal structure of LyPls-PLD exhibited a TIM barrel fold assigned to glycerophosphocholine phosphodiesterase, a member of glycerophosphodiester phosphodiesterase. LyPls-PLD possesses a hydrophobic cleft for the binding of the aliphatic chain of the substrate. In the structure of the F211L mutant, Met232 and Tyr258 form a "small lid" structure that stabilizes the binding of the aliphatic chain of the substrate. In contrast, F211 may inhibit small lid formation in the wild-type structure. LysoPAF possesses a flexible aliphatic chain; therefore, a small lid is effective for stabilizing the substrate during catalytic reactions.

Discovery and Biological Characterization of PRMT5:MEP50 Protein-Protein Interaction Inhibitors.

Protein arginine methyltransferase 5 (PRMT5) is a master epigenetic regulator and an extensively validated therapeutic target in multiple cancers. Notably, PRMT5 is the only PRMT that requires an obligate cofactor, methylosome protein 50 (MEP50), to function. We developed compound 17, a novel small-molecule PRMT5:MEP50 protein-protein interaction (PPI) inhibitor, after initial virtual screen hit identification and analogue refinement. Molecular docking indicated that compound 17 targets PRMT5:MEP50 PPI by displacing the MEP50 W54 burial into a hydrophobic pocket of the PRMT5 TIM barrel. In vitro analysis indicates IC50 < 500 nM for prostate and lung cancer cells with selective, specific inhibition of PRMT5:MEP50 substrate methylation and target gene expression, and RNA-seq analysis suggests that compound 17 may dysregulate TGF-β signaling. Compound 17 provides a proof of concept in targeting PRMT5:MEP50 PPI, as opposed to catalytic targeting, as a novel mechanism of action and supports further preclinical development of inhibitors in this class.

Structural and functional insights into the glycoside hydrolase family 30 xylanase of the rumen bacterium Ruminococcus flavefaciens

The family 30 glycoside hydrolase (RfGH30) from Ruminococcus flavefaciens expressing xylanase activity was homology modeled. The structure revealed a (β/α)8 TIM barrel topology along with an associated ‘side β-structure’. Secondary structure analysis by circular dichroism displayed 28.09% α-helices and 21.02% β-strands, which corroborated with the prediction results. Multiple Sequence Alignment and structure superposition of RfGH30 with its homologue indicate that Glu200 and Glu302 are the catalytic residues displaying a retaining-type of catalysis. Molecular Dynamics (MD) simulation of RfGH30 confirmed the structural compactness and steadiness of modeled structure. Molecular docking studies showed maximum binding affinity with xylobiose that was corroborated by the total binding Gibbs free energy of -160.5 kJ/mol calculated by g_mmpbsa tool. MD simulation of RfGH30-xylobiose confirmed the structural specificity of catalytic residues and its increased stability in presence of ligand. Small angle X-ray scattering (SAXS) analysis of RfGH30 confirmed its monodispersed, fully folded and elongated structure in solution form. Dummy atom model revealed monomeric form of RfGH30 at 2.8 mg/ml, while at 4 mg/ml as dimer. Dynamic light scattering analysis of RfGH30 showed monodispersity and increase in hydrodynamic radius (Rh) from 2.0 to 4.0 mg/mL, displaying possible dimerization of RfGH30 at 4.0 mg/mL as corroborated by SAXS analysis. The lowered zeta potential of RfGH30 at 4 mg/mL also indicated the formation of a dimer.

In-Depth Characterization of Debranching Type I Pullulanase from Priestia koreensis HL12 as Potential Biocatalyst for Starch Saccharification and Modification

Pullulanase is an effective starch debranching enzyme widely used in starch saccharification and modification. In this work, the biochemical characteristics and potential application of a new type I pullulanase from Priestia koreensis HL12 (HL12Pul) were evaluated and reported for the first time. Through in-depth evolutionary analysis, HL12Pul was classified as type I pullulanase belonging to glycoside hydrolase family 13, subfamily 14 (GH13_14). HL12Pul comprises multi-domains architecture, including two carbohydrate-binding domains, CBM68 and CBM48, at the N-terminus, the TIM barrel structure of glycoside hydrolase family 13 (GH13) and C-domain. Based on sequence analysis and experimental cleavage profile, HL12Pul specifically hydrolyzes only α-1,6 glycosidic linkage-rich substrates. The enzyme optimally works at 40 °C, pH 6.0, with the maximum specific activity of 181.14 ± 3.55 U/mg protein and catalytic efficiency (kcat/Km) of 49.39 mL/mg·s toward pullulan. In addition, HL12Pul worked in synergy with raw starch-degrading α-amylase, promoting raw cassava starch hydrolysis and increasing the sugar yield by 2.9-fold in comparison to the α-amylase alone in a short reaction time. Furthermore, HL12Pul effectively produces type III-resistant starch (RSIII) from cassava starch with a production yield of 70%. These indicate that HL12Pul has the potential as a biocatalyst for starch saccharification and modification.

Crystal structure of Methanococcus jannaschii dihydroorotase.

In this paper, we report the structural analysis of dihydroorotase (DHOase) from the hyperthermophilic and barophilic archaeon Methanococcus jannaschii. DHOase catalyzes the reversible cyclization of N-carbamoyl-l-aspartate to l-dihydroorotate in the third step of de novo pyrimidine biosynthesis. DHOases form a very diverse family of enzymes and have been classified into types and subtypes with structural similarities and differences among them. This is the first archaeal DHOase studied by x-ray diffraction. Its structure and comparison with known representatives of the other subtypes help define the structural features of the archaeal subtype. The M. jannaschii DHOase is found here to have traits from all subtypes. Contrary to expectations, it has a carboxylated lysine bridging the two Zn ions in the active site, and a long catalytic loop. It is a monomeric protein with a large β sandwich domain adjacent to the TIM barrel. Loop 5 is similar to bacterial type III and the C-terminal extension is long.

Structural and Functional Analysis of the Pyridoxal Phosphate Homeostasis Protein YggS from Fusobacterium nucleatum.

Pyridoxal 5′-phosphate (PLP) is the active form of vitamin B6, but it is highly reactive and poisonous in its free form. YggS is a PLP-binding protein found in bacteria and humans that mediates PLP homeostasis by delivering PLP to target enzymes or by performing a protective function. Several biochemical and structural studies of YggS have been reported, but the mechanism by which YggS recognizes PLP has not been fully elucidated. Here, we report a functional and structural analysis of YggS from Fusobacterium nucleatum (FnYggS). The PLP molecule could bind to native FnYggS, but no PLP binding was observed for selenomethionine (SeMet)-derivatized FnYggS. The crystal structure of FnYggS showed a type III TIM barrel fold, exhibiting structural homology with several other PLP-dependent enzymes. Although FnYggS exhibited low (<35%) amino acid sequence similarity with previously studied YggS proteins, its overall structure and PLP-binding site were highly conserved. In the PLP-binding site of FnYggS, the sulfate ion was coordinated by the conserved residues Ser201, Gly218, and Thr219, which were positioned to provide the binding moiety for the phosphate group of PLP. The mutagenesis study showed that the conserved Ser201 residue in FnYggS was the key residue for PLP binding. These results will expand the knowledge of the molecular properties and function of the YggS family.

Enabling Role of Ligand-Driven Conformational Changes in Enzyme Evolution.

Many enzymes that show a large specificity in binding the enzymatic transition state with a higher affinity than the substrate utilize substrate binding energy to drive protein conformational changes to form caged substrate complexes. These protein cages provide strong stabilization of enzymatic transition states. Using part of the substrate binding energy to drive the protein conformational change avoids a similar strong stabilization of the Michaelis complex and irreversible ligand binding. A seminal step in the development of modern enzyme catalysts was the evolution of enzymes that couple substrate binding to a conformational change. These include enzymes that function in glycolysis (triosephosphate isomerase), the biosynthesis of lipids (glycerol phosphate dehydrogenase), the hexose monophosphate shunt (6-phosphogluconate dehydrogenase), and the mevalonate pathway (isopentenyl diphosphate isomerase), catalyze the final step in the biosynthesis of pyrimidine nucleotides (orotidine monophosphate decarboxylase), and regulate the cellular levels of adenine nucleotides (adenylate kinase). The evolution of enzymes that undergo ligand-driven conformational changes to form active protein-substrate cages is proposed to proceed by selection of variants, in which the selected side chain substitutions destabilize a second protein conformer that shows compensating enhanced binding interactions with the substrate. The advantages inherent to enzymes that incorporate a conformational change into the catalytic cycle provide a strong driving force for the evolution of flexible protein folds such as the TIM barrel. The appearance of these folds represented a watershed event in enzyme evolution that enabled the rapid propagation of enzyme activities within enzyme superfamilies.

An artificial metalloprotein with metal-adaptive coordination sites and Ni-dependent quercetinase activity

Engineering non-native metal active sites into proteins using canonical amino acids offers many advantages but is hampered by significant challenges. The TIM barrel protein, imidazole glycerol phosphate synthase from the hyperthermophilic organism Thermotoga maritima (tHisF), is well-suited for the construction of artificial metalloenzymes by this approach. To this end, we have generated a tHisF variant (tHisFEHH) with a Glu/His/His motif for metal ion coordination. Crystal structures of ZnII:tHisFEHH and NiII:tHisFEHH reveal that both metal ions bind to the engineered histidines. However, the two metals bind at distinct sites with different geometries, demonstrating the adaptability of tHisF. Only ZnII additionally ligates the Glu residue and adopts a tetrahedral geometry. The pseudo-octahedral NiII site comprises the two His and a native Ser residue. NiII:tHisFEHH catalyzes the oxidative cleavage of the flavanols quercetin and myricetin, providing an unprecedented example of an artificial metalloprotein with quercetinase activity.

Crystal structure of chloroplast fructose-1,6-bisphosphate aldolase from the green alga Chlamydomonas reinhardtii

The Calvin-Benson cycle fixes carbon dioxide into organic triosephosphates through the collective action of eleven conserved enzymes. Regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco-mediated carboxylation, requires two lyase reactions catalyzed by fructose-1,6-bisphosphate aldolase (FBA). While cytoplasmic FBA has been extensively studied in non-photosynthetic organisms, functional and structural details are limited for chloroplast FBA encoded by oxygenic phototrophs. Here we determined the crystal structure of plastidial FBA from the unicellular green alga Chlamydomonas reinhardtii (Cr). We confirm that CrFBA folds as a TIM barrel, describe its catalytic pocket and homo-tetrameric state. Multiple sequence profiling classified the photosynthetic paralogs of FBA in a distinct group from non-photosynthetic paralogs. We mapped the sites of thiol- and phospho-based post-translational modifications known from photosynthetic organisms and predict their effects on enzyme catalysis.

Structural Investigation of AlsA, a Radical S‐adenosylmethionine Enzyme Involved in the Biosynthesis of the Oxetane‐containing Herbicide Albucidin

Herbicides are a class of molecules commonly employed to maintain high crop yields by targeting weeds, a particularly damaging class of pests in agriculture. Albucidin is a novel herbicide found in Streptomyces albus subsp. Chlorinus NRRL B‐24108 that shows a suspected new mode of action. Albucidin is a nucleoside analogue containing an oxetane moiety similar to the antiviral compound oxetanocin‐A (OXT‐A). OXT‐A is the only other known naturally occurring oxetane‐containing nucleoside analogue and has a biosynthetic pathway with two key enzymes: OxsA and OxsB. OsxB is a B12‐dependent radical S‐adenosylmethionine (RS) enzyme and catalyzes formation of the oxetane ring via an oxidative ring contraction. A similar reaction has been proposed to be catalyzed by the B12‐dependent RS enzyme AlsB in the biosynthetic pathway of albucidin; however, the two biosynthetic pathways appear to diverge at this point as well. While OXT‐A is formed upon subsequent reduction and OxsA catalyzed dephosphorylation of the OxsB reaction product, the chemistry leading to albucidin is much less clear. In particular, one carbon must be eliminated, and AlsA, which is a biotin synthase‐like RS enzyme also encoded in the albucidin biosynthetic cluster, has been proposed to participate in this reaction. AlsA is most closely related to BioB‐like enzymes and demonstrates high sequence similarity with the canonical TIM barrel domain; however, AlsA also contains an N‐terminal domain of about 100 amino acids that shares minimal sequence homology to other RS enzymes with known structures. Herein, recent developments in the structural analysis of AlsA using X‐ray crystallography are reported.

Understanding the Molecular Mechanism of PLCε Regulation and G Protein‐Mediated Activation

Cardiovascular disease is the leading cause of death in the world. The phospholipase C (PLC) family of enzymes, in particular the PLCε subfamily, are essential for normal cardiovascular function. PLCε hydrolyzes phosphatidylinositol phosphates at cellular membranes, producing inositol phosphates (IPx) and diacylglycerol (DAG). These crucial secondary messengers activate multiple downstream pathways, including cardiac contractility and the expression of hypertrophic genes. In the cardiovascular system, PLCε is regulated through direct interactions with the RhoA and Rap1A small GTPases, which in turn are activated downstream of G protein‐coupled receptors (GPCRs). RhoA is reported to activate PLCε at the plasma membrane, whereas Rap1A translocates and activates PLCε at the perinuclear membrane. However, the elements within PLCε that regulate basal activity and membrane association have not been fully identified. Similarly, the domains involved in Rap1A versus RhoA binding, activation and translocation to different subcellular membranes have not been mapped. In this work, we use a structure‐guided approach, together with cell‐based activity assays, epifluorescence, and confocal microscopy to identify the roles of PLCε regulatory elements and domains in basal activity, subcellular localization, and regulation by RhoA and Rap1A GTPases. Functional studies show N‐ and C‐terminal regulatory domains of PLCε dictate its location within the cell, and contribute differently to basal and G protein‐dependent activity. We also show that regulatory insertions within the catalytic TIM barrel, including the X–Y linker and Y‐box, aid in interfacial activation and membrane association. These studies provide much needed insights into the molecular determinants of PLCε that regulate its localization and activity in cells, which is critical for elucidating its roles in cardiovascular function.

Structural Determinants of Sugar Alcohol Biosynthesis in Plants: The Crystal Structures of Mannose-6-Phosphate and Aldose-6-Phosphate Reductases.

Sugar alcohols are major photosynthetic products in plant species from the Apiaceae and Plantaginaceae families. Mannose-6-phosphate reductase (Man6PRase) and aldose-6-phosphate reductase (Ald6PRase) are key enzymes for synthesizing mannitol and glucitol in celery (Apium graveolens) and peach (Prunus persica), respectively. In this work, we report the first crystal structures of dimeric plant aldo/keto reductases (AKRs), celery Man6PRase (solved in the presence of mannonic acid and NADP+) and peach Ald6PRase (obtained in the apo form). Both structures displayed the typical TIM barrel folding commonly observed in proteins from the AKR superfamily. Analysis of the Man6PRase holo form showed that residues putatively involved in the catalytic mechanism are located close to the nicotinamide ring of NADP+, where the hydride transfer to the sugar phosphate should take place. Additionally, we found that Lys48 is important for the binding of the sugar phosphate. Interestingly, the Man6PRase K48A mutant had a lower catalytic efficiency with mannose-6-phosphate but a higher catalytic efficiency with mannose than the wild type. Overall, our work sheds light on the structure-function relationships of important enzymes to synthesize sugar alcohols in plants.

Structure and Function Insight of the α-Glucosidase QsGH13 From Qipengyuania seohaensis sp. SW-135.

The α-glucosidases play indispensable roles in the metabolic mechanism of organism, prevention, and treatment of the disease, and sugar hydrolysis, and are widely used in chemical synthesis, clinical diagnosis, and other fields. However, improving their catalytic efficiency and production to meet commercial demand remains a huge challenge. Here we detected a novel GH13 family α-glucosidase, QsGH13, from the deep-sea bacterium Qipengyuania seohaensis sp. SW-135. QsGH13 is highly substrate specific and only hydrolyzes sugars containing alpha-1,4 glucoside bonds. For example, its enzymatic activity for p-nitrophenyl-α-D-glucopyranoside was 25.41 U/mg, and the Km value was 0.2952 ± 0.0322 mM. The biochemical results showed that the optimum temperature of QsGH13 is 45°C, the optimum pH is 10.0, and it has excellent biological characteristics such as alkali resistance and salt resistance. The crystal structure of QsGH13 was resolved with a resolution of 2.2 Å, where QsGH13 is composed of a typical TIM barrel catalytic domain A, a loop-rich domain B, and a conserved domain C. QsGH13 crystal belonged to the monoclinic space group P212121, with unit-cell parameters a = 58.816 Å, b = 129.920 Å, c = 161.307 Å, α = γ = β = 90°, which contains two monomers per asymmetric unit. The β → α loop 4 of QsGH13 was located above catalytic pocket. Typical catalytic triad residues Glu202, Asp266, and Glu329 were found in QsGH13. The biochemical properties and structural analysis of QsGH13 have greatly improved our understanding of the catalytic mechanism of GH13 family. This study provides new ideas to broaden the application of α-glucosidase in alcohol fermentation, glycolysis, and other industries.

Double mutations far from the active site affect cold activity in an Antarctic halophilic β-galactosidase.

The Antarctic haloarchaeon, Halorubrum lacusprofundi, contains a polyextremophilic family 42 β-galactosidase, which we are using as a model for cold-active enzymes. Divergent amino acid residues in this 78 kDa protein were identified through comparative genomics and hypothesized to be important for cold activity. Six amino acid residues were previously mutated and five were shown by steady-state kinetic analysis to have altered temperature-dependent catalytic activity profiles via effects on Km and/or kcat compared to the wild-type enzyme. In this follow-up study, double-mutated enzymes were constructed and tested for temperature effects, including two new tandem residue pairs (N180T/A181T and T383A/S384A), and pairwise combination of the single residue mutations (N251D, F387L, I476V, and V482L). All double-mutated enzymes were found to be more catalytically active at moderate and/or less active at colder temperatures than wild-type, with both Km and kcat effects observed for the two tandem mutations. For pairwise combinations, a Km effect was seen when the surface exposed F387L mutation located in a domain A TIM barrel α helix 19 Å from the active site was combined with two internal residues, N251D or V482L. When another surface exposed mutation I476V located in a coiled region of domain B 25 Å from the active site was paired with N251D or V482L, a kcat effect was observed. These results indicate that temperature-dependent kinetic effects may be complex and subtle and are mediated by a combination of a small number of residues distant from the active site via changes to the hydration shell and/or perturbation of internal packing.

De Novo Design of a Highly Stable Ovoid TIM Barrel: Unlocking Pocket Shape towards Functional Design.

The ability to finely control the structure of protein folds is an important prerequisite to functional protein design. The TIM barrel fold is an important target for these efforts as it is highly enriched for diverse functions in nature. Although a TIM barrel protein has been designed de novo, the ability to finely alter the curvature of the central beta barrel and the overall architecture of the fold remains elusive, limiting its utility for functional design. Here, we report the de novo design of a TIM barrel with ovoid (twofold) symmetry, drawing inspiration from natural beta and TIM barrels with ovoid curvature. We use an autoregressive backbone sampling strategy to implement our hypothesis for elongated barrel curvature, followed by an iterative enrichment sequence design protocol to obtain sequences which yield a high proportion of successfully folding designs. Designed sequences are highly stable and fold to the designed barrel curvature as determined by a 2.1 Å resolution crystal structure. The designs show robustness to drastic mutations, retaining high melting temperatures even when multiple charged residues are buried in the hydrophobic core or when the hydrophobic core is ablated to alanine. As a scaffold with a greater capacity for hosting diverse hydrogen bonding networks and installation of binding pockets or active sites, the ovoid TIM barrel represents a major step towards the de novo design of functional TIM barrels.

Profiles of Natural and Designed Protein-Like Sequences Effectively Bridge Protein Sequence Gaps: Implications in Distant Homology Detection.

Sequence-based approaches are fundamental to guide experimental investigations in obtaining structural and/or functional insights into uncharacterized protein families. Powerful profile-based sequence search methods rely on a sequence space continuum to identify non-trivial relationships through homology detection. The computational design of protein-like sequences that serve as "artificial linkers" is useful in identifying relationships between distant members of a structural fold. Such sequences act as intermediates and guide homology searches between distantly related proteins. Here, we describe an approach that represents natural intermediate sequences and designed protein-like sequences as HMM (Hidden Markov Models) profiles, to improve the sensitivity of existing search methods. Searches made within the "Profile database" were shown to recognize the parent structural fold for 90% of the search queries at query coverage better than 60%. For 1040 protein families with no available structure, fold associations were made through searches in the database of natural and designed sequence profiles. Most of the associations were made with the Alpha-alpha superhelix, Transmembrane beta-barrels, TIM barrel, and Immunoglobulin-like beta-sandwich folds. For 11 domain families of unknown functions, we provide confident fold associations using the profiles of designed sequences and a consensus from other fold recognition methods. For two DUFs (Domain families of Unknown Functions), we performed detailed functional annotation through comparisons with characterized templates of families of known function.

A newly introduced salt bridge cluster improves structural and biophysical properties of de novo TIM barrels.

Protein stability can be fine‐tuned by modifying different structural features such as hydrogen‐bond networks, salt bridges, hydrophobic cores, or disulfide bridges. Among these, stabilization by salt bridges is a major challenge in protein design and engineering since their stabilizing effects show a high dependence on the structural environment in the protein, and therefore are difficult to predict and model. In this work, we explore the effects on structure and stability of an introduced salt bridge cluster in the context of three different de novo TIM barrels. The salt bridge variants exhibit similar thermostability in comparison with their parental designs but important differences in the conformational stability at 25°C can be observed such as a highly stabilizing effect for two of the proteins but a destabilizing effect to the third. Analysis of the formed geometries of the salt bridge cluster in the crystal structures show either highly ordered salt bridge clusters or only single salt bridges. Rosetta modeling of the salt bridge clusters results in a good prediction of the tendency on stability changes but not the geometries observed in the three‐dimensional structures. The results show that despite the similarities in protein fold, the salt bridge clusters differently influence the structural and stability properties of the de novo TIM barrel variants depending on the structural background where they are introduced.

Functional and structural characterization of a GH3 β-N-acetylhexosaminidase from Akkermansia muciniphila involved in mucin degradation

Akkermansia muciniphila is a probiotic that colonizes the outer layer of intestinal mucus and is negatively associated with metabolic disorders. Amuc_2109 protein, a β-N-acetylhexosaminidase from A. muciniphila, may be involved in the degradation of mucins and is associated with intestinal health. Here, we reported the crystal structure of Amuc_2109, which belongs to the GH family 3 enzymes and fell into the canonical (α/β)8 TIM barrel structure with GlcNAc bound to the active center. Biochemical assay characterization of Amuc_2109 revealed that Amuc_2109 is a GlcNAc-specific glycosidase active over a wide temperature and pH range, reflecting the survival advantage of Amuc_2109 in the intestinal environment. Our structural and biochemical results will contribute to the understanding of the catalytic mechanism of the GH3 β-N-acetylhexosaminidase and help to gain insight into the molecular mechanism of complex carbohydrate utilization and restoration of the intestinal barrier in A. muciniphila.

Experimental and in silico studies of competitive inhibition of family GH10 Aspergillus fumigatus xylanase A by Oryza sativa xylanase inhibitor protein

The family GH10 Aspergillus fumigatus xylanase A (AfXylA10) gene, afxyla10 was cloned and recombinantly expressed in Pichia pastoris X33. The optimum temperature and pH of reAfXylA10 was 53 °C and 7.0, and Mn2+ remarkably activated the catalytic activity. The recombinant Oryza sativa xylanase inhibitor protein, rePOsXIP significantly inhibited reAfXylA10 with inhibition constant (Ki) of 177.94 nM via competitive inhibition and decreased the concentration of hydrolysate from beechwood xylan. Optimal inhibition of rePOsXIP on reAfXylA10 occurred at 45 °C for 40 min. The fluorescence of reAfXylA10 was statically quenched by rePOsXIP, indicating the formation of reAfXylA10-rePOsXIP complex during their interaction. Furthermore, molecular dynamics (MD) simulations were performed to obtain the detailed information on enzyme-inhibitor interaction. The binding free energy (ΔG) of AfXylA10-OsXIP complex was −30 ± 9 kcal/mol by MM-PBSA calculation, and the α-7 helix of OsXIP anchored in the catalytic cleft of AfXylA10 by competition with the xylan substrate. K239OsXIP stably interacted with the catalytic site E140AfXylA10 through hydrogen bond and vdW interaction. Intermolecular hydrogen bonds T104AfXylA10/V99AfXylA10-Q5OsXIP, R256AfXylA10-E235OsXIP, D155AfXylA10-Y243OsXIP and D145AfXylA10-R194OsXIP on the upper of the TIM barrel were essential for strengthening the stability of complex. Therefore, these non-covalent interactions (NCI) played key role in the interaction between AfXylA10 and OsXIP.