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Alpha amylase belonging to starch hydrolyzing enzymes has significant contributions to different industrial processes. The enzyme production through recombinant DNA technology faces certain challenges related to their expression, solubility and purification, which can be overcome through fusion tags. This study explored the influence of SUMO, a protein tag reported to enhance the solubility and stability of target proteins when fused to the N-terminal of the catalytic domain of amylase from Pyrococcus abyssi (PaAD). The insoluble expression of PaAD in E. coli was overcome when the enzyme was expressed in a fusion state (S-PaAD) and culture was cultivated at 18 °C. Moreover, the activity of S-PaAD increased by 1.5-fold as compared to that of PaAD. The ligand binding and enzyme activity assays against different substrates demonstrated that it was more active against 1 % glycogen and amylopectin. The analysis of the hydrolysates through HPLC demonstrated that the enzyme activity is mainly amylolytic, producing longer oligosaccharides as the major end product. The secondary structure analyses by temperature ramping in CD spectroscopy and MD simulation demonstrated the enzymes in the free, as well as fusion state, were stable at 90 °C. The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various foods, feed, textiles, detergents, pharmaceuticals, and many industrial applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Endogenous phospholipase A 2 (PLA 2 ) plays an important role in phospholipids degradation during cured meat products manufacturing. The present study was undertaken to reveal more information about the endogenous PLA 2 in muscles and its role in degradation of intramuscular phospholipids. With the catalytic domain of pork calcium-independent PLA 2 (iPLA 2 cd), impacts of physic-chemical factors on the activity were investigated and substrate specificity of the enzyme were tested respectively. The optimum temperature and pH of pork iPLA 2 cd were 40 °C and 7.5, respectively. The iPLA 2 cd could be stimulated by adequate contents of NaCl and ATP, and inhibited by CaCl 2 and NaNO 2 . For native phospholipids, the iPLA 2 cd was of a little higher affinity towards phosphatidylcholine (PC) than phosphatidylethanolamine (PE), phosphoserine (PS) and phosphatidylinositol (PI). The iPLA 2 cd could preferentially hydrolyze peroxidized PC over the native PC. The results would help better understand the degradation of phospholipids and the role played by endogenous enzymes during meat products manufacturing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To facilitate the eukaryotic repriming pathway of DNA damage tolerance, PrimPol synthesises de novo oligonucleotide primers downstream of polymerase-stalling obstacles. These primers enable replicative polymerases to resume synthesis and ensure the timely completion of DNA replication. Initiating synthesis de novo requires the coordination of single-stranded DNA, initiating nucleotides, and metal ions within PrimPol's active site to catalyze the formation of the first phosphodiester bond. Here we examine the interactions between human PrimPol's catalytic domain, nucleotides, and DNA template during each of the various catalytic steps to determine the 'choreography' of primer synthesis, where substrates bind in an ordered manner. Our findings show that the ability of PrimPol to conduct de novo primer synthesis is underpinned by a network of stabilising interactions between the enzyme, template, and nucleotides, as we previously observed for related primase CRISPR-Associated Prim-Pol (CAPP). Together, these findings establish a detailed model for the initiation of DNA synthesis by human PrimPol, which appears highly conserved., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Recent research certified that DOT1L and its mutations represented by R231Q were potential targets for the treatment of lung cancer. Herein, a series of adenosine-containing derivatives were identified with DOT1LR231Q inhibition through antiproliferation assay and Western blot analysis in the H460R231Q cell. The most promising compound 37 significantly reduced DOT1LR231Q mediated H3K79 methylation and effectively inhibited the proliferation, self-renewal, migration, and invasion of lung cancer cell lines at low micromolar concentrations. The cell permeability and cellular target engagement of 37 were verified by both CETSA and DARTS assays. In the H460R231Q OE cell-derived xenograft (CDX) model, 37 displayed pronounced tumor growth inhibition after intraperitoneal administration at 20 mg/kg dose for 3 weeks (TGI = 54.38%), without obvious toxicities. A pharmacokinetic study revealed that 37 possessed tolerable properties (t1/2 = 1.93 ± 0.91 h, F = 97.2%) after intraperitoneal administration in rats. Mechanism study confirmed that 37 suppressed malignant phenotypes of lung cancer carrying R231Q gain-of-function mutation via the MAPK/ERK signaling pathway. Moreover, analysis of the binding modes between molecules and DOT1LWT/R231Q proteins put forward the “Induced-fit” allosteric model in favor to the discovery of potent DOT1L candidates.

Phenolic compounds are typical organic pollutants which cause severe human health problems due to their teratogenesis, carcinogenesis, neurotoxicity, immunotoxicity and endocrine disruption. Natural laccase is a multicopper oxidase existing in bacteria, plants, and insects, which can accelerate the transformation of phenolic compounds to their less hazardous oxidized products under mild conditions without harmful byproducts. Despite eco-environmentally friendly property of laccase, it still faces constraints of widespread application attribute to its high cost, complex preparation, and vulnerability. Therefore, exploring laccase mimics with high catalytic activity attracts a lot of attention and endeavors. In this research, copper-based nanozymes were prepared with coordination of copper ions and imidazole for mimicking the active sites of natural laccase via solvothermal method. The obtained Cu-based (Cu-Im) nanozymes exhibited multiple redox valence states of Cu and laccase-mimicking coordination structures, which endow Cu-Im with high laccase-like activity. During the process of catalytic oxidation reactions, singlet oxygen and superoxide anions generated from oxygen. Encouraged by the catalytic property, Cu-Im was utilized in degradation and detection of phenolic and catecholamine compounds. The catalytic degradation of compounds by Cu-Im showed good conversion and substrate versatility, which can be used as a kind of potential materials for phenolic pollutant degradation and remediation. Simultaneously, colorimetric sensors of phenols and catecholamines based on Cu-Im in solution system and POCT pad platform were constructed which indicated wide linear range and low limit of detection for both detection strategies. The Cu-Im-based sensor was a promising method for sensitive, fast, convenient, and qualitative-quantitative colorimetric analysis of phenols and catecholamines. The outcomes of this research elucidate Cu-Im is a satisfactory substitute for natural laccase, which will have broad application prospects in laccase-related fields, such as environmental recovery, pollution monitoring, and diagnosis of neurological diseases etc., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Konjac oligosaccharide (KOS) exhibits various biological activities, and hyperthermophilic β-glucanases offer many advantages for KOS production from konjac glucanmannan (KGM). In this study, a novel β-glucanase, EG003, belonging to the glycosyl hydrolase (GH) subfamily 5_1, was predicted from the genome of the a Thermus strain. The recombinant EG003 and its catalytic domain, EG003A, were successfully expressed and characterized. EG003A displayed maximum activity at 95 °C and pH 8.0, with a specific activity of 1047.6 U/mg and retained approximately 50 % activity after 6 h at 90 °C. The enzyme exhibited both β-1,4-glucanase and β-1,3-1,4-glucanase activity with KGM and sodium carboxymethylcellulose (CMC), lichenan and oat β-glucan as substrates. Degree of polymerization (DP) 3 was the major oligosaccharide from the hydrolysis of KGM, while DP3 and DP4 were predominant products from the hydrolysis of oat β-glucan. Molecular docking analyses revealed that the catalytic mechanism of EG003A is consistent with those of other reported GH5_1 β-glucanases. Additionally, the viscosity of 500 mL solution of 1 % KGM decreased rapidly from 31,193 mPa.s to 4.50 mPa.s in 3 min with 30 U EG003A. This study provides an efficient hyperthermophilic β-glucanase with promising application for KOS production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier B.V.)

Poly((R)-3-hydroxybutyrate) (PHB) is a microbial biopolymer widely used in commercial biodegradable plastics. PHB degradation in cell is catalyzed by PHB depolymerase (PhaZ), which hydrolyzes the polyester into mono- and/or oligomeric (R)-3-hydroxylbutyrates (3HB). A novel intracellular PhaZ from Bacillus thuringiensis (BtPhaZ) was identified for potential applications in polymer biodegradation and 3HB production. Herein, we present the crystal structure of BtPhaZ at 1.42-Å resolution, making the first crystal structure for an intracellular PhaZ. BtPhaZ comprises a canonical α/β hydrolase catalytic domain and a unique α-helical cap domain. Despite lacking sequence similarity, BtPhaZ shares high structural homology with many α/β hydrolase members, exhibiting a similar active-site architecture. Alongside the most conserved superfamily signature, several new conserved signatures have been identified, contributing not only to the formations of the Ser-His-Asp catalytic triad and the oxyanion hole but also to the active-site conformation. The putative P-1 subsite appears to have limited space for accommodating only one 3HB-monomer, which may provide an explanation why the major hydrolytic product for BtPhaZ is monomeric form. Furthermore, a cluster of solvent-exposed hydrophobic residues in the helical cap domain forms an adsorption site for polymer-binding. Detailed structural comparisons reveal that various PhaZs employ distinct residues for the biopolymer-binding and hydrolysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A highly conserved second-sphere active site αSer residue in nitrile hydratase (NHase), that forms a hydrogen bond with the axial metal-bound water molecule, was mutated to Ala, Asp, and Thr, in the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) and to Ala and Thr in the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase). All five mutants were successfully purified; metal analysis via ICP-AES indicated that all three Co-type PtNHase mutants were in their apo-form while the Fe-type αSer117Ala and αSer117Thr mutants contained 85 and 50 % of their active site Fe(III) ions, respectively. The k cat values obtained for the PtNHase mutant enzymes were between 0.03 ± 0.01 and 0.2 ± 0.02 s -1 amounting to <0.8 % of the k cat value observed for WT PtNHase. The Fe-type ReNHase mutants retained some detectable activity with k cat values of 93 ± 3 and 40 ± 2 s -1 for the αSer117Ala and αSer117Thr mutants, respectively, which is ∼5 % of WT ReNHase activity towards acrylonitrile. UV-Vis spectra coupled with EPR data obtained on the ReNHase mutant enzymes showed subtle changes in the electronic environment around the active site Fe(III) ions, consistent with altering the hydrogen bonding interaction with the axial water ligand. X-ray crystal structures of the three PtNHase mutant enzymes confirmed the mutation and the lack of active site metal, while also providing insight into the active site hydrogen bonding network. Taken together, these data confirm that the conserved active site αSer residue plays an important catalytic role but is not essential for catalysis. They also confirm the necessity of the conserved second-sphere αSer residue for the metalation process and subsequent post-translational modification of the α-subunit in Co-type NHases but not Fe-type NHases, suggesting different mechanisms for the two types of NHases. SYNOPSIS: A strictly conserved active site αSer residue in both Co- and Fe-type nitrile hydratases was mutated. This αSer residue was found to play an important catalytic function, but is not essential. In Co-type NHases, it appears to be essential for active site maturation, but not in Fe-type NHases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Oxygen-tolerant [NiFe] hydrogenases are valuable blueprints for the activation and evolution of molecular hydrogen under application-relevant conditions. Vibrational spectroscopic techniques play a key role in the investigation of these metalloenzymes. For instance, resonance Raman spectroscopy has been introduced as a site-selective approach for probing metal-ligand coordinates of the [NiFe] active site and FeS clusters. Despite its success, this approach is still challenged by a limited number of detectable active-site states - due to missing resonance enhancement or intrinsic light sensitivity - and difficulties in their assignment. Utilizing two oxygen-tolerant [NiFe] hydrogenases as model systems, we illustrate how these challenges can be met by extending excitation and detection wavelength regimes in resonance Raman spectroscopic studies. Specifically, we observe that this technique does not only probe low-frequency metal-ligand vibrations but also high-frequency intra-ligand modes of the diatomic CO/CN - ligands at the active site of [NiFe] hydrogenases. These reporter vibrations are routinely probed by infrared absorption spectroscopy, so that direct comparison of spectra from both techniques allows an unambiguous assignment of states detected by resonance Raman spectroscopy. Moreover, we find that a previously undetected state featuring a bridging hydroxo ligand between Ni and Fe can be probed using higher excitation wavelengths, as photoconversion occurring at lower wavelengths is avoided. In summary, this study expands the applicability of resonance Raman spectroscopy to hydrogenases and other complex metalloenzymes by introducing new strategies for probing and assigning redox-structural states of the active site., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Staphylococcus aureus (S. aureus) is one of the common causes of implant associated biofilm infections and their biofilms are resistant to antibiotics. S. aureus amidase (AM) protein, a cell wall hydrolase that cleaves the amide bond between N-acetylmuramic acid and L-alanine residue of the stem peptide, is several fold over-expressed under biofilm conditions. Previous studies demonstrated an autolysin mutant in S. aureus that lacks the AM protein, is highly impaired in biofilm development. We carried out a structure-based small molecule design using the crystal structure of AM protein catalytic domain to identify inhibitors that can block amidase activity and therefore inhibits S. aureus biofilm formation. Sequential virtual screening followed by pharmacokinetic analysis and bioassay studies filtered 25 small molecules from different databases. Two compounds from the SPECS database, SPECS-1 and SPECS-2, were selected based on the best docking score and minimum biofilm inhibitory concentration towards S. aureus biofilms. SPECS-1 and SPECS-2 were further tested for their structural/energetic stability in complex with the AM protein using molecular dynamics simulation and MM-GBSA techniques. In vitro, biofilm inhibition studies on different surfaces confirmed that treatment with SPECS-1 and SPECS-2 at a concentration of 250 μg/ml exhibited significant prevention and disruption of S. aureus biofilms. Finally, the in vitro anti-biofilm activities of these two compounds were validated against Methicillin-resistant S. aureus clinical isolates. We concluded that the discovered compounds SPECS-1 and SPECS-2 are safe and exhibit biofilm preventive and disruption activity for inhibiting the S. aureus biofilms and hence can be used to treat implant-associated biofilm infections., Competing Interests: Declaration of competing interest The authors declare no competing interests of any type that can influence the work done in this article., (Copyright © 2023. Published by Elsevier B.V.)

Ribonuclease A is a major hydrolyzing enzyme involved in the hydrolysis of RNA. The crystals of bovine pancreatic RNase A (bpRNase A) were grown at pH 5.5. The effect of sodium cyanide on bpRNase A was assessed by adding it directly to the crystal containing well. Treating the crystals of bpRNase A with sodium cyanide resulted in the displacement of the sulfate ion from the active site of bpRNase A, while the additional sulfate ion, bound to Ala-4, remained unaffected. The addition of sodium cyanide to bpRNase A crystals did not show change in the secondary structure elements of the enzyme. This study was conducted to check the effect of cyanide on bpRNase A crystals and to displace sulfate ion from its active site., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

To fully understand enzymatic dynamics, it is essential to explore the complete conformational space of a biological catalyst. The catalytic mechanism of the nickel-dependent urease, the most efficient enzyme known, holds significant relevance for medical, pharmaceutical, and agro-environmental applications. A critical aspect of urease function is the conformational change of a helix-turn-helix motif that covers the active site cavity, known as the mobile flap. This motif has been observed in either an open or a closed conformation through X-ray crystallography studies and has been proposed to stabilize the coordination of a urea molecule to the essential dinuclear Ni(II) cluster in the active site, a requisite for subsequent substrate hydrolysis. This study employs cryo-electron microscopy (cryo-EM) to investigate the transient states within the conformational space of the mobile flap, devoid of the possible constraints of crystallization conditions and solid-state effects. By comparing two cryo-EM structures of Sporosarcina pasteurii urease, one in its native form and the other inhibited by N-(n-butyl) phosphoric triamide (NBPTO), we have unprecedently identified an intermediate state between the open and the catalytically efficient closed conformation of the helix-turn-helix motif, suggesting a role of its tip region in this transition between the two states., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Giancarlo Tria reports financial support was provided by European Union. Luca Mazzei reports equipment, drugs, or supplies was provided by European Union. Cianci Michele reports a relationship with Elettra Sincrotrone Trieste SCpA that includes: consulting or advisory. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Helicobacter pylori deoxyuridine triphosphate nucleotidohydrolase (HpdUTPase) is a key enzyme in the synthesis of the thymidine nucleotide pathway. It catalyzes the hydrolysis of dUTP to dUMP and releases pyrophosphate. This enzyme has been shown to be essential in several pathogenic organisms. Here, we have determined the crystal structures of HpdUTPase in complex with α, β-imido dUTP (non-hydrolyzable substrate analog) and apo-state at resolution of 2 Å and 2.5 Å respectively. The flexible c terminal end of HpdUTPase which is not observed in apo-state structure and becomes ordered in the complex structure, suggesting its role in forming active site and substrate interaction. The Isothermal titration calorimetry (ITC) experiments reveal that hydrolysis of dUTP is an exothermic reaction with K m  = 35.0 ± 0.19 μM and the k cat  = 1.20 ± 0.19 s -1 . The ITC studies combined with MD simulations for all other nucleotides (dATP.dGTP, dCTP and dTTP) show that the active site of HpdUTPase strangely can also accommodate dATP. The structural comparison with the host (human) dUTPases reveals critical differences in substrate binding affinity of the active site of HpdUTPase. The detailed study suggests that the dATP binds in the active site of HpdUTPase making the number of preferable hydrogen bonds and shows activity with Km of 47 ± 2.4 μM., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Samudrala Gourinath reports financial support was provided by India Ministry of Science & Technology Department of Biotechnology. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Transition state (TS) stabilization by enzymes greatly accelerates catalytic reactions. For some enzymes, the TS complex has entropy higher than enzyme substrate (ES) complex. But the origin of favorable entropy remains unclear. In this work, we studied the mechanism of Bacillus Circulans xylanase (BCX) 11 catalyzed o-nitrophenyl β-xylobioside (ONPX 2 ) glycoside hydrolysis. The catalytic reaction exhibits a positive activation entropy, and an increase in ionic strength leads to a decrease in entropy without affecting the activation free energy, indicating that the entropy is predominantly influenced by electrostatic forces. Moreover, NMR measurements of electrostatic attractions within the active site demonstrate a positive entropy, aligning with molecular dynamics (MD) simulations showing that electrostatic interactions contribute to the entropic stabilization of the TS complex. These findings suggest that the positive entropy primarily originates from alterations in electrostatic interactions due to the formation of the oxocarbenium ion at C 1 in the TS. Differences of electrostatic interactions in ES and TS modify hydrogen bonding of surrounding residues in the active site which causes their side chain dynamics and thus conformational entropy changes. Residues critical for the positive activation entropy are identified. A new BCX mutant with an increased activation entropy and catalytic activity is found., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In consideration of life in extreme environments, the effects of hydrostatic pressure on proteins at the atomic level have drawn substantial interest. Large deviations of temperature and pressure from ambient conditions can shift the free energy landscape of proteins to reveal otherwise lowly populated structural states and even promote unfolding. We report the crystal structure of the heme-containing peroxidase, cytochrome c peroxidase (CcP) at 1.5 and 3.0 kbar and make comparisons to structures determined at 1.0 bar and cryo-temperatures (100 K). Pressure produces anisotropic changes in CcP, but compressibility plateaus after 1.5 kbar. CcP responds to pressure with volume declines at the periphery of the protein where B-factors are relatively high but maintains nearly intransient core structure, hydrogen bonding interactions and active site channels. Changes in active-site solvation and heme ligation reveal pressure sensitivity to protein-ligand interactions and a potential docking site for the substrate peroxide. Compression at the surface affects neither alternate side-chain conformers nor B-factors. Thus, packing in the core, which resembles a crystalline solid, limits motion and protects the active site, whereas looser packing at the surface preserves side-chain dynamics. These data demonstrate that conformational dynamics and packing densities are not fully correlated in proteins and that encapsulation of cofactors by the polypeptide can provide a precisely structured environment resistant to change across a wide range of physical conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Sialic acid esterase (SIAE) catalyzes the removal of O-acetyl groups from sialic acids found on cell surface glycoproteins to regulate cellular processes such as B cell receptor signalling and apoptosis. Loss-of-function mutations in SIAE are associated with several common autoimmune diseases including Crohn's, ulcerative colitis, and arthritis. To gain a better understanding of the function and regulation of this protein, we determined crystal structures of SIAE from three mammalian homologs, including an acetate bound structure. The structures reveal that the catalytic domain adopts the fold of the SGNH hydrolase superfamily. The active site is composed of a catalytic dyad, as opposed to the previously reported catalytic triad. Attempts to determine a substrate-bound structure yielded only the hydrolyzed product acetate in the active site. Rigid docking of complete substrates followed by molecular dynamics simulations revealed that the active site does not form specific interactions with substrates, rather it appears to be broadly specific to accept sialoglycans with diverse modifications. Based on the acetate bound structure, a catalytic mechanism is proposed. Structural mapping of disease mutations reveals that most are located on the surface of the enzyme and would only cause minor disruptions to the protein fold, suggesting that these mutations likely affect binding to other factors. These results improve our understanding of SIAE biology and may aid in the development of therapies for autoimmune diseases and cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Extradiol dioxygenase Tcu3516 is a home-sourced enzyme demonstrating potent aromatic phenol degradation capacity. To add to the advantageous modifications inside active cavity, this work reported a novel strategy to engineer rarely concerned non-regular structures around the entrance towards the active site at the bottom of cavity. Three structures, Loop region 1 (Loop1: Met173-Arg185), Loop region 2 (Loop2: Ala201-Val212) and C-terminal (C-tail: His290-Lys306) were therefore identified through structural flexibility analysis. Highly rigid prolines within the structures were mutated into smaller alanine, glycine, or serine to improve structural flexibilities; while only P183S on Loop1 showed 3-fold activity enhancement vs the WT when subjected to cleavage of mono-cyclic catechol analogues. The analysis of Root Mean Square Fluctuation showed that P183S presents certain enhancement on Loop1 flexibility without dramatic changes of other domains. Furthermore, the synergetic effects from mutation P183S and cavity-based mutations V186L, V212N and D285A were evaluated by characterizing combinatorial mutants. Temperature dependence and thermostability of the combined mutants showed a more flexible catalytic domain without sacrificing structural integrity and stability. k cat value of P183S/V186L (SL) towards monocyclic catechols significantly surpasses any other combinatorial mutants around Tcu3516 active sites. Moreover, the synergetic effects on conformational plasticity were analyzed by molecular dynamic simulations to shed light into the interplay between structural changes and catalytic performance., Competing Interests: Declaration of competing interest All authors declare no competing financial interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Novel classes of antibiotics are needed to improve the resilience of the healthcare system to antimicrobial resistance (AMR), including vancomycin resistance. vanA gene cluster is a cause of vancomycin resistance. This gene cluster is transferred and spreads vancomycin resistance from Enterococcus spp. to Staphylococcus aureus. Therefore, novel antibacterial agents are required to combat AMR, including vanA-type vancomycin resistance. Serine hydroxymethyltransferase (SHMT) is a key target of antibacterial agents. However, the specific binding mechanisms of SHMT inhibitors remain unclear. Detailed structural information will contribute to understanding these mechanisms. In this study, we found that (+)-SHIN-2, the first in vivo active inhibitor of human SHMT, is strongly bound to the Enterococcus faecium SHMT (efmSHMT). Comparison of the crystal structures of apo- and (+)-SHIN-2-boud efmSHMT revealed that (+)-SHIN-2 stabilized the active site loop of efmSHMT via hydrogen bonds, which are critical for efmSHMT inhibition. Additionally, (+)-SHIN-2 formed hydrogen bonds with serine, forming the Schiff's base with pyridoxal 5'-phosphate, which is a co-factor of SHMT. Furthermore, (+)-SHIN-2 exerted biostatic effects on vancomycin-susceptible and vanA-type vancomycin-resistant E. faecium in vitro, indicating that SHMT inhibitors do not induce cross-resistance to vanA-type vancomycin. Overall, these findings can aid in the design of novel SHMT inhibitors to combat AMR, including vancomycin resistance., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Recognizing the critical need to elucidate the molecular determinants of this selectivity offers a pathway to engineer enzymes with broader and more versatile catalytic capabilities. Through integrated methods including phylogenetic analysis, molecular docking, and structural analysis, we identified a pivotal amino acid residue, αTrp116, linking the substrate binding pocket and the active site of a NHase from Pseudonocardia thermophila JCM 3095 (PtNHase). This residue acts as a crucial determinant of substrate specificity within the NHase enzyme. The mutant αW116R modified the substrate specificity of PtNHase, significantly enhancing its catalytic efficiency towards aromatic substrates. The catalytic activity for aromatic compounds such as 3-Cyanopyridine was 14-fold that of the wild-type, whereas its activity for aliphatic substrates diminished to one-sixth. MD simulations revealed that replacing αTrp116 with Arg allowed aromatic nitrile substrates to achieve more favorable conformations within the active site. Based on the mutant αW116R, we further constructed a combinatorial variant Pt-4, tailored for aromatic substrates, which exhibited an enzyme activity 50 times that of the wild-type. These results highlight the critical influence of amino acid residues in the enzyme's active site on substrate specificity and offer fresh perspectives and approaches for the evolution of enzymes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Inulosucrases are enzymes capable of synthesizing inulin polymers using sucrose as the main substrate. The enzymatic activity relies on the catalytic triad within the active site and residues responsible for substrate recognition and orientation, termed carbohydrate-binding subsites. This study investigates the role of specific residues within the catalytic cavity of a truncated version of IslA4 in enzymatic catalysis. Mutants at residues S425, L499, A602, R618, F619, Y676, Y692, and R696 were constructed and characterized. Characterization results, and in silico structural comparison with other fructansucrases, reveal these residues' functional significance in catalysis. Residue S425 belongs to subsite -1; residues R618 and Y692 are part of subsite +1, and residue R696 belongs to subsites +1 and +2. Residues L499 and A602 are support residues; the former favors the formation of the fructosyl-enzyme intermediate, while the latter stabilizes the acid/base catalyst during catalysis. Residues Y676 and F619 may participate in stabilizing residues at -1/+1 subsites. This study represents the first comprehensive exploration of the structural determinants essential for enzymatic function in the inulosucrase of Leuconostoc citreum, and proposes the identity of residues involved in the -1 to +2 subsites., Competing Interests: Declaration of competing interest Ingrid Mercado Del Rio reports financial support was provided by Consejo Nacional de Humanidades, Ciencia y Tecnología (CONAHCyT). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Some sulfur-oxidizing bacteria playing an important role in global geochemical cycles utilize thiocyanate as the sole source of energy and nitrogen. In these bacteria the process of thiocyanate into cyanate conversion is mediated by thiocyanate dehydrogenases - a recently discovered family of copper-containing enzymes with the three‑copper active site unique among the other copper proteins. To get a deeper insight into the structure and molecular mechanism of action of thiocyanate dehydrogenases we isolated, purified, and comprehensively characterized an enzyme from the bacterium Pelomicrobium methylotrophicum. High-resolution crystal structures of the thiocyanate dehydrogenase in the free state and in the complexes with the transition state analog, thiourea, and the closest substrate analog, selenocyanate, unveiled the fine details of molecular events occurring at the enzyme active site. During the reaction thiocyanate dehydrogenase undergoes profound conformational change that affects the position of the constituent copper ions and results in the activation of the attacking water molecule. The structure of the enzyme complex with the selenium atom bridged in-between two copper ions was obtained representing an important transient intermediate. Structures of the complexes with inhibitors supplemented with quantum chemical calculations clarify the role of copper ions and refine molecular mechanism of catalysis by thiocyanate dehydrogenase., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Despite the availability of high resolution crystal structure of the catalytic domain of human carbonic anhydrase IX (denoted as HCA IX-c), its solution structure and functional dynamics are mostly unknown. In this article, we report classical molecular dynamics (MD) simulation studies on the structure and non-reactive dynamics of dimeric HCA IX-c (d-HCA IX-c, with chains A and B) in water and compare them to each chain simulated in water as a monomeric solute (mA/mB). While the average solution structures appear to be largely similar in all systems, d-HCA IX-c shows significant deviations from mA and mB in terms of non-reactive reorganization of the active site needed to initiate the rate determining proton transfer step between a zinc-bound water and one of the sidechain N-atoms of a distal histidine residue (His-64). First, 2−6 water molecules are found to form fluctuating hydrogen bonded networks that would serve as proton paths across the active site. A minimally frustrated core of each monomer in the dimeric protein is found to be associated with a significantly higher likelihood of forming complete proton transfer pathways mediated by 2 water molecules synchronously at the two active sites hosted by the monomeric chains. The relative stability and kinetics of transition between two major sidechain rotamers of His-64 are also affected by dimerization. The sidechain rotation of His-64 is found to involve at least one transition as slow as 105s−1 in chain A of d-HCA IX-c thereby rendering it slower than the actual rate determining proton transfer step. This observation indicates an important deviation from the widely accepted mechanism of catalysis by HCAs.

Histone lysine demethylase (KDM), AlkB homolog (ALKBH), and Ten-Eleven Translocation (TET) proteins are members of the 2-Oxoglutarate (2OG) and ferrous iron-dependent oxygenases, each of which harbors a catalytic domain centered on a double-stranded β-helix whose topology restricts the regions directly involved in substrate binding. However, they have different catalytic functions, and the deeply structural biological reasons are not yet clear. In this review, the catalytic domain features of the three protein families are summarized from both sequence and structural perspectives. The construction of the phylogenetic tree and comparison of the structure show ten relatively conserved β-sheets and three key regions with substantial structural differences. We summarize the relationship between three key regions of remarkable differences and the substrate compatibility of the three protein families. This review facilitates research into substrate-selective inhibition and bioengineering by providing new insights into the catalytic domains of KDM, ALKBH, and TET proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In the global fight against the COVID-19 pandemic caused by the highly transmissible SARS-CoV-2 virus, the search for potent medications is paramount. With a focused investigation on the SARS-CoV-2 papain-like protease (PL pro ) as a promising therapeutic target due to its pivotal role in viral replication and immune modulation, the catalytic triad of PL pro comprising Cys111, His272, and Asp286, highlights Cys111 as an intriguing nucleophilic center for potential covalent bonds with ligands. The detailed analysis of the binding site unveils crucial interactions with both hydrophobic and polar residues, demonstrating the structural insights of the cavity and deepening our understanding of its molecular landscape. The sequence of PL pro among variants of concern (Alpha, Beta, Gamma, Delta and Omicron) and the recent variant of interest, JN.1, remains conserved with no mutations at the active site. Moreover, a thorough exploration of apo, non-covalently bound, and covalently bound PL pro conformations exposes significant conformational changes in loop regions, offering invaluable insights into the intricate dynamics of ligand-protein complex formation. Employing strategic in silico medication repurposing, this study swiftly identifies potential molecules for target inhibition. Within the domain of covalent docking studies and molecular dynamics, using reported inhibitors and clinically tested molecules elucidate the formation of stable covalent bonds with the cysteine residue, laying a robust foundation for potential therapeutic applications. These details not only deepen our comprehension of PL pro inhibition but also play a pivotal role in shaping the dynamic landscape of COVID-19 treatment strategies., Competing Interests: Declaration of Competing Interest None, (Copyright © 2024. Published by Elsevier Ltd.)

S-omeprazole and R-rabeprazole are important proton pump inhibitors (PPIs) used for treating peptic disorders. They can be biosynthesized from the corresponding sulfide catalyzed by Baeyer-Villiger monooxygenases (BVMOs). During the development of BVMOs for target sulfoxide preparation, stereoselectivity and overoxidation degree are important factors considered most. In the present study, LnPAMO-Mu15 designed previously and TtPAMO from Thermothelomyces thermophilus showed high (S)- and (R)-configuration stereoselectivity respectively towards thioethers. TtPAMO was found to be capable of oxidating omeprazole sulfide (OPS) and rabeprazole sulfide (RPS) into R-omeprazole and R-rabeprazole respectively. However, the overoxidation issue existed and limited the application of TtPAMO in the biosynthesis of sulfoxides. The structural mechanisms for adverse stereoselectivity between LnPAMO-Mu15 and TtPAMO towards OPS and the overoxidation of OPS by TtPAMO were revealed, based on which, TtPAMO was rationally designed focused on the flexibility of loops near catalytic sites. The variant TtPAMO-S482Y was screened out with lowest overoxidation degree towards OPS and RPS due to the decreased flexibility of catalytic center than TtPAMO. The success in this study not only proved the rationality of the overoxidation mechanism proposed in this study but also provided hints for the development of BVMOs towards thioether substrate for corresponding sulfoxide preparation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Glycoside hydrolase family 91 (GH91) inulin fructotransferase (IFTases) enables biotransformation of fructans into sugar substitutes for dietary intervention in metabolic syndrome. However, the catalytic mechanism underlying the sequential biodegradation of inulin remains unelusive during the biotranformation of fructans. Herein we present the crystal structures of IFTase from Arthrobacter aurescens SK 8.001 in apo form and in complexes with kestose, nystose, or fructosyl nystose, respectively. Two kinds of conserved noncatalytic binding regions are first identified for IFTase-inulin interactions. The conserved interactions of substrates were revealed in the catalytic center that only contained a catalytic residue E205. A switching scaffold was comprised of D194 and Q217 in the catalytic channel, which served as the catalytic transition stabilizer through side chain displacement in the cycling of substrate sliding in/out the catalytic pocket. Such features in GH91 contribute to the catalytic model for consecutive cutting of substrate chain as well as product release in IFTase, and thus might be extended to other exo-active enzymes with an enclosed bottom of catalytic pocket. The study expands the current general catalytic principle in enzyme-substrate complexes and shed light on the rational design of IFTase for fructan biotransformation., Competing Interests: Declaration of competing interest There is no conflict of interests regarding the publication of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

MltG, positioned within the inner membrane of bacteria, functions as a lytic transglycosylase (LT) essential for integrating into the cell wall by cleaving the newly synthesized glycan strand, emphasizing its critical involvement in bacterial cell wall biosynthesis and remodeling. Current study reported the first structure of MltG family of LT. We have elucidated the structure of MltG from Acinetobacter baumannii (abMltG), a formidable superbug renowned for its remarkable antibiotic resistance. Our structural and biochemical investigations unveiled the presence of a flexible peptidoglycan (PG)-binding domain (PGD) within MltG family, which exists as a monomer in solution. Furthermore, we delineated the putative active site of abMltG via a combination of structural analysis and sequence comparison. This discovery enhances our comprehension of the transglycosylation process mediated by the MltG family, offering insights that could inform the development of novel antibiotics tailored to combat A. baumannii., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The purple acid phosphatase was purified from 5.9-fold to apparent homogeneity from Anagelis arvensis seeds using SP-Sephadex C-50 and Sephadex G-100 chromatography. The results of residual activity tests conducted using different temperature ranges (50-70 °C) were calculated as the activation energy (E d  = 72 kJ/mol), enthalpy (69.31 ≤ (ΔH° ≤ 69.10 kJ/mol), entropy (-122.48 ≤ ΔS° ≤ -121.13 J/mol·K), and Gibbs free energy (108.87 ≤ ΔG° ≤ 111.25 kJ/mol) of the enzyme irreversible denaturation. These thermodynamic parameters indicate that this novel PAP is highly thermostable and may be significant for use in industrial applications. However, it may be confirmed by stopped-flow measurements that this substitution produces a chromophoric Fe 3+ site and a Pi-substrate interaction that is about ten times faster. Additionally, these data show that phenyl phosphate hydrolysis proceeds more rapidly in metal form of A. arvensis PAP than the creation of a μ-1,3 phosphate complex. The Fe 3+ site in the native Fe 3+ -Mn 2+ derivative interacts with it at a faster rate than in the Fe 3+ -Fe 2+ form. This is most likely caused by a network of hydrogen bonds between the first and second coordination spheres. This suggests that the choice of metal ions plays a significant role in regulating the activity of this enzyme., Competing Interests: Declaration of competing interest The author has no conflict of interest exits in the submission of this manuscript and the authors have complied with journal ethical requirements. Moreover, manuscript is approved by all authors as well as all the authorities acknowledged for publication., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Human glycosyltransferases (GTs) play crucial roles in glycan biosynthesis, exhibiting diverse domain architectures. This study explores the functional diversity of "add-on" domains within human GTs, using data from the AlphaFold Protein Structure Database. Among 215 annotated human GTs, 74 contain one or more add-on domains in addition to their catalytic domain. These domains include lectin folds, fibronectin type III, and thioredoxin-like domains and contribute to substrate specificity, oligomerization, and consequent enzymatic activity. Notably, certain GTs possess dual enzymatic functions due to catalytic add-on domains. The analysis highlights the importance of add-on domains in enzyme functionality and disease implications, such as congenital disorders of glycosylation. This comprehensive overview enhances our understanding of GT domain organization, providing insights into glycosylation mechanisms and potential therapeutic targets., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Pullulanases are important starch-debranching enzymes that mainly hydrolyze the α-1,6-glycosidic linkages in pullulan, starch, and oligosaccharides. Nevertheless, their practical applications are constrained because of their poor activity and low thermostability. Moreover, the trade-off between activity and thermostability makes it challenging to simultaneously improve them. In this study, an engineered pullulanase was developed through reshaping the active-site tunnel and engineering the surface lysine residues using the pullulanase from Pyrococcus yayanosii CH1 (Pul PY2 ). The specific activity of the engineered pullulanase was increased 3.1-fold, and thermostability was enhanced 1.8-fold. Moreover, the engineered pullulanase exhibited 11.4-fold improvement in catalytic efficiency (k cat /K m ). Molecular dynamics simulations demonstrated an anti-correlated movement around the entrance of active-site tunnel and stronger interactions between the surface residues in the engineered pullulanase, which would be beneficial to the activity and thermostability improvement, respectively. The strategies used in this study and dynamic evidence for insight into enzyme performance improvement may provide guidance for the activity and thermostability engineering of other enzymes., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Recent research certified that DOT1L and its mutations represented by R231Q were potential targets for the treatment of lung cancer. Herein, a series of adenosine-containing derivatives were identified with DOT1L R231Q inhibition through antiproliferation assay and Western blot analysis in the H460 R231Q cell. The most promising compound 37 significantly reduced DOT1L R231Q mediated H3K79 methylation and effectively inhibited the proliferation, self-renewal, migration, and invasion of lung cancer cell lines at low micromolar concentrations. The cell permeability and cellular target engagement of 37 were verified by both CETSA and DARTS assays. In the H460 R231Q OE cell-derived xenograft (CDX) model, 37 displayed pronounced tumor growth inhibition after intraperitoneal administration at 20 mg/kg dose for 3 weeks (TGI = 54.38%), without obvious toxicities. A pharmacokinetic study revealed that 37 possessed tolerable properties ( t 1/2  = 1.93 ± 0.91 h, F  = 97.2%) after intraperitoneal administration in rats. Mechanism study confirmed that 37 suppressed malignant phenotypes of lung cancer carrying R231Q gain-of-function mutation via the MAPK/ERK signaling pathway. Moreover, analysis of the binding modes between molecules and DOT1L WT/R231Q proteins put forward the "Induced-fit" allosteric model in favor to the discovery of potent DOT1L candidates., Competing Interests: The authors have no conflicts of interest to declare., (© 2024 The Authors.)

Engineering biocatalysts with enhanced stereoselectivity is highly desirable, and active-site loop dynamics play an important role in its regulation. However, knowledge of their precise roles in catalysis and evolution is limited. Here, we used the strategy of Rosetta enzyme design combined molecular dynamic simulations (MDs) to reprogram the landscapes of the key active-site loop dynamics of the carbonyl reductase LfSDR1 to improve stereoselectivity. The key flexible loop in the active site showed the potential to regulate the catalytic properties. A library of virtual variants was produced using the Rosetta design and assessed dynamic effect of the loop with the aid of MDs. A potential candidate was obtained with significant stereoselectivity (ee > 99 %) compared to the wild-type (ee = 42 %) without loss of catalytic activity or thermostability. The molecular basis of the catalytic property enhancement was flanked by MDs, which revealed the role of the G92L mutation in regulating loop dynamics to stabilize the environment of the active site. Finally, a series of the challenge bulky substrate derivatives were assessed using the G92L variant, and all showed improved stereoselectivity ee > 99 %. This study provides novel insights for improving stereoselectivity through rational engineering of the loop dynamics of biocatalysts., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2024. Published by Elsevier B.V.)

Glycoside hydrolase family 5 (GH5) encompasses enzymes with several different activities, including endo-1,4-β-mannosidases. These enzymes are involved in mannan degradation, and have a number of biotechnological applications, such as mannooligosaccharide prebiotics production, stain removal and dyes decolorization, to name a few. Despite the importance of GH5 enzymes, only a few members of subfamily 7 were structurally characterized. In the present work, biochemical and structural characterization of Bacillus licheniformis GH5 mannanase, BlMan5&#95;7 were performed and the enzyme cleavage pattern was analyzed, showing that BlMan5&#95;7 requires at least 5 occupied subsites to perform efficient hydrolysis. Additionally, crystallographic structure at 1.3 Å resolution was determined and mannoheptaose (M7) was docked into the active site to investigate the interactions between substrate and enzyme through molecular dynamic (MD) simulations, revealing the existence of a - 4 subsite, which might explain the generation of mannotetraose (M4) as an enzyme product. Biotechnological application of the enzyme in stain removal was investigated, demonstrating that BlMan5&#95;7 addition to washing solution greatly improves mannan-based stain elimination., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Summary: Peptidyl arginine deiminases (PADIs) catalyze protein citrullination, a post-translational conversion of arginine to citrulline. The most widely expressed member of this family, PADI2, regulates cellular processes that impact several diseases. We hypothesized that we could gain new insights into PADI2 function through a systematic evolutionary and structural analysis. Here, we identify 20 positively selected PADI2 residues, 16 of which are structurally exposed and maintain PADI2 interactions with cognate proteins. Many of these selected residues reside in non-catalytic regions of PADI2. We validate the importance of a prominent loop in the middle domain that encompasses PADI2 L162, a residue under positive selection. This site is essential for interaction with the transcription elongation factor (P-TEFb) and mediates the active transcription of the oncogenes c-MYC, and CCNB1, as well as impacting cellular proliferation. These insights could be key to understanding and addressing the role of the PADI2 c-MYC axis in cancer progression.

α-L-Rhamnosidase has attracted lots of attention due to its industrial potential applications. The applicability of α-L-rhamnosidase, however, was limited by their low ligand affinity on the industrial scale. In order to improve the affinity of α-L-rhamnosidase for industrial use, we investigated the variation of its affinity by amino acid replacement. Particularly, the enzyme affinity of a α-L-rhamnosidase from Aspergillus niger JMU-TS528 (rRha1) was measured with the semi-conservative amino acid (homology between 30% -80%) replaced. As a result, the enzyme affinity of the two mutants, R404S and N578D, were increased by 1.45-fold and 2.3-fold, respectively, showing that these two mutants could be the promising candidates for industrial use. To test if these mutations bring negative effect on the enzyme properties, we also determined the other enzymatic properties of these mutants and showed no negative effect. To understand the improvement of enzyme affinity, the conformational flexibility of (α/α) 6 -barrel catalytic domain were examined by molecular dynamics (MD) simulation, and demonstrated that the conformations of these mutants are more flexible, which could influence the affinity of substrates to the enzyme and hence the enzyme activity. This work not only enhanced the enzyme affinity of a α-L-rhamnosidase, making rRha1 a promising candidate for industrial processes, but also provided an effective technical strategy for improving affinity of other enzymes., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

N-terminal autoprocessing from its polyprotein precursor enables creating the mature-like stable dimer interface of SARS-CoV-2 main protease (MPro), concomitant with the active site oxyanion loop equilibrium transitioning to the active conformation (E*) and onset of catalytic activity. Through mutagenesis of critical interface residues and evaluating noncovalent inhibitor (ensitrelvir, ESV) facilitated dimerization through its binding to MPro, we demonstrate that residues extending from Ser1 through Glu14 are critical for dimerization. Combined mutations G11A, E290A and R298A (MPro™) restrict dimerization even upon binding of ESV to monomeric MPro™ with an inhibitor dissociation constant of 7.4 ± 1.6 µM. Contrasting the covalent inhibitor NMV or GC373 binding to monomeric MPro, ESV binding enabled capturing the transition of the oxyanion loop conformations in the absence of a reactive warhead and independent of dimerization. Characterization of complexes by room-temperature X-ray crystallography reveals ESV bound to the E* state of monomeric MPro as well as an intermediate approaching the inactive state (E). It appears that the E* to E equilibrium shift occurs initially from G138-F140 residues, leading to the unwinding of the loop and formation of the 3 10 -helix. Finally, we describe a transient dimer structure of the MPro precursor held together through interactions of residues A5-G11 with distinct states of the active sites, E and E*, likely representing an intermediate in the autoprocessing pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Clostridioides difficile endolysin (Ecd09610) consists of an unknown domain at its N terminus, followed by two catalytic domains, a glucosaminidase domain and endopeptidase domain. X-ray structure and mutagenesis analyses of the Ecd09610 catalytic domain with glucosaminidase activity (Ecd09610CD53) were performed. Ecd09610CD53 was found to possess an α-bundle-like structure with nine helices, which is well conserved among GH73 family enzymes. The mutagenesis analysis based on X-ray structures showed that Glu405 and Asn470 were essential for enzymatic activity. Ecd09610CD53 may adopt a neighboring-group mechanism for a catalytic reaction in which Glu405 acted as an acid/base catalyst and Asn470 helped to stabilize the oxazolinium ion intermediate. Structural comparisons with the newly identified Clostridium perfringens autolysin catalytic domain (AcpCD) in the P1 form and a zymography analysis demonstrated that AcpCD was 15-fold more active than Ecd09610CD53. The strength of the glucosaminidase activity of the GH73 family appears to be dependent on the depth of the substrate-binding groove., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Structure-function relationships are key to understanding enzyme mechanisms, controlling enzyme activities, and designing biocatalysts. Here, we investigate the functions of arginine residues in the active sites of pyridoxal-5'-phosphate (PLP)-dependent non-canonical d-amino acid transaminases, focusing on the analysis of a transaminase from Haliscomenobacter hydrossis. Our results show that the tandem of arginine residues R28* and R90, which form the conserved R-[RK] motif in non-canonical d-amino acid transaminases, not only facilitates effective substrate binding but also regulates the catalytic properties of PLP. Non-covalent interactions between residues R28*, R90, and Y147 strengthen the hydrogen bond between Y147 and PLP, thereby maintaining the reactivity of the cofactor. Next, the R90 residue contributes to the stability of the holoenzyme. Finally, the R90I substitution induces structural changes that lead to substrate promiscuity, as evidenced by the effective binding of substrates with and without the α-carboxylate group. This study sheds light on the structural determinants of the activity of non-canonical d-amino acid transaminases. Understanding the structural basis of the active site plasticity in the non-canonical transaminase from H. hydrossis, which is characterized by effective conversion of d-amino acids and α-keto acids, may help to tailor it for industrial applications., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Glutathione reductase (GR) is a two dinucleotide binding domain flavoprotein (tDBDF) that catalyzes the reduction of glutathione disulfide to glutathione coupled to the oxidation of NADPH to NADP + . An interesting feature of GR and other tDBDFs is the presence of a lysine residue (Lys-66 in human GR) at the active site, which interacts with the flavin group, but has an unknown function. To better understand the role of this residue, the dynamics of GR was studied using molecular dynamics simulations, and the reaction mechanism of FAD reduction by NADPH was studied using QM/MM molecular modeling. The two possible protonation states of Lys-66 were considered: neutral and protonated. Molecular dynamics results suggest that the active site is more structured for neutral Lys-66 than for protonated Lys-66. QM/MM modeling results suggest that Lys-66 should be in its neutral state for a thermodynamically favorable reduction of FAD by NADPH. Since the reaction is unfavorable with protonated Lys-66, the reverse reaction (the reduction of NADP + by FADH - ) is expected to take place. A phylogenetic analysis of various tDBDFs was performed, finding that an active site lysine is present in different the tDBDFs enzymes, suggesting that it has a conserved biological role. Overall, these results suggest that the protonation state of the active site lysine determines the energetics of the reaction, controlling its reversibility., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Histidine residues 44 and 48 in yeast alcohol dehydrogenase (ADH) bind to the coenzymes NAD(H) and contribute to catalysis. The individual H44R and H48Q substitutions alter the kinetics and pH dependencies, and now the roles of other ionizable groups in the enzyme were studied in the doubly substituted H44R/H48Q ADH. The substitutions make the enzyme more resistant to inactivation by diethyl pyrocarbonate, modestly improve affinity for coenzymes, and substantially decrease catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The pH dependencies for several kinetic parameters are shifted from pK values for wild-type ADH of 7.3-8.1 to values for H44R/H48Q ADH of 8.0-9.6, and are assigned to the water or alcohol bound to the catalytic zinc. It appears that the rate of binding of NAD + is electrostatically favored with zinc-hydroxide whereas binding of NADH is faster with neutral zinc-water. The pH dependencies of catalytic efficiencies (V/E t K m ) for ethanol oxidation and acetaldehyde reduction are similarly controlled by deprotonation and protonation, respectively. The substitutions make an enzyme that resembles the homologous horse liver H51Q ADH, which has Arg-47 and Gln-51 and exhibits similar pK values. In the wild-type ADHs, it appears that His-48 (or His-51) in the proton relay systems linked to the catalytic zinc ligands modulate catalytic efficiencies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Bryce V Plapp reports financial support was provided by US Public Health Services. The corresponding author has served as managing editor for special issues of Chem.-Biol. Interactions. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Northern corn leaf blight caused by Setosphaeria turcica is a major fungal disease responsible for significant reductions in maize yield worldwide. Eukaryotic type 2A protein phosphatase (PP2A) influences growth and virulence in a number of pathogenic fungi, but little is known about its roles in S. turcica. Here, we functionally characterized S. turcica StPP2A-C, which encodes the catalytic C subunit of StPP2A. StPP2A-C deletion slowed colony growth, conidial germination, and appressorium formation but increased conidiation, melanin biosynthesis, glycerol content, and disease lesion size on maize. These effects were associated with expression changes in genes related to calcium signaling, conidiation, laccase activity, and melanin and glycerol biosynthesis, as well as changes in intra- and extracellular laccase activity. A pull-down screen for candidate StPP2A-c interactors revealed an interaction between StPP2A-c and StLac1. Theoretical modeling and yeast two-hybrid experiments confirmed that StPP2A-c interacted specifically with the copper ion binding domain of StLac1 and that Cys267 of StPP2A-c was required for this interaction. StPP2A-C expression thus appears to promote hyphal growth and reduce pathogenicity in S. turcica, at least in part by altering melanin synthesis and laccase activity; these insights may ultimately support the development of novel strategies for biological management of S. turcica., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Natural product bulgecin A potentiates the activity of β-lactam antibiotics by inhibition of three lytic transglycosylases in Pseudomonas aeruginosa, of which MltD is one. MltD exhibits both endolytic and exolytic reactions in the turnover of the cell-wall peptidoglycan and tolerates the presence or absence of stem peptides in its substrates. The present study reveals structural features of the multimodular MltD, presenting a catalytic module and four cell-wall-binding LysM modules that account for these attributes. Three X-ray structures are reported herein for MltD that disclose one unpredicted LysM module tightly attached to the catalytic domain, whereas the other LysM modules are mobile, and connected to the catalytic domain through long flexible linkers. The formation of crystals depended on the presence of bulgecin A. The expansive active-site cleft is highlighted by the insertion of a helical region, a hallmark of the family 1D of lytic transglycosylases, which was mapped out in a ternary complex of MltD:bulgecinA:chitotetraose, revealing at the minimum the presence of eight subsites (from -4 to +4, with the seat of reaction at subsites -1 and + 1) for binding of sugars of the substrate for the endolytic reaction. The mechanism of the exolytic reaction is revealed in one of the structures, showing how the substrate's terminal anhydro-NAM moiety could be sequestered at subsite +2. Our results provide the structural insight for both the endolytic and exolytic activities of MltD during cell-wall-turnover events., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Carbohydrate-binding module (CBM) family 91 is a novel module primarily associated with glycoside hydrolase (GH) family 43 enzymes. However, our current understanding of its function remains limited. PphXyl43B is a β-xylosidase/α-L-arabinofuranosidase bifunctional enzyme from physcomitrellae patens XB belonging to the GH43&#95;11 subfamily and containing CBM91 at its C terminus. To fully elucidate the contributions of the CBM91 module, the truncated proteins consisting only the GH43&#95;11 catalytic module (rPphXyl43B-dCBM91) and only the CBM91 module (rCBM91) of PphXyl43B were constructed, respectively. The result showed that rPphXyl43B-dCBM91 completely lost hydrolysis activity against both p-nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside; it also exhibited significantly reduced activity towards xylobiose, xylotriose, oat spelt xylan and corncob xylan compared to the control. Thus, the CBM91 module is crucial for the β-xylosidase/α-L-arabinofuranosidase activities in PphXyl43B. However, rCBM91 did not exhibit any binding capability towards corncob xylan. Structural analysis indicated that CBM91 of PphXyl43B might adopt a loop conformation (residues 496-511: ILSDDYVVQSYGGFFT) to actively contribute to the catalytic pocket formation rather than substrate binding capability. This study provides important insights into understanding the function of CBM91 and can be used as a reference for analyzing the action mechanism of GH43&#95;11 enzymes and their application in biomass energy conversion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

PARP15, or ARTD7, is an enzyme carrying out mono-ADP-ribosylation and regulating activities of a range of cellular proteins. This enzyme belongs to the family of the poly(ADP-ribose) polymerases (PARPs), which comprises of proteins with various potential disease indications. Due to their involvement in a number of cellular processes and important role in DNA repair and regulation, PARPs have been considered attractive therapeutic targets over the past few years. The pursuit of small molecule PARP inhibitors has resulted in several FDA approved drugs for multiple cancers so far. As the use of PARP inhibitors as drug scaffolds is actively explored recently, there is increasing interest in the design of selective inhibitors based on the structural features of the PARP proteins. Here, we solved high-resolution crystal structures of the human PARP15 catalytic domain in complex with three marketed drugs of PARP inhibitors, which includes compounds 3-AB, iniparib and niraparib. The structures reported here contribute to our understanding of the ligand binding modes and structural features in the PARP15 catalytic domain, which can be employed to guide the rational design of selective inhibitors of PARPs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Small ubiquitin-related modifier (SUMO) proteins are efficiently used to target the soluble expression of various difficult-to-express proteins in E. coli. However, its utilization in large scale protein production is restricted by the higher cost of Ulp, which is required to cleave SUMO fusion tag from protein-of-interest to generate an authentic N-terminus. This study identified and characterized two novel SUMO proteases i.e., Ulp1 and Ulp2 from Schizosaccharomyces pombe. Codon-optimized gene sequences were cloned and expressed in E. coli. The sequence and structure of SpUlp1 and SpUlp2 catalytic domains were deduced using bioinformatics tools. Protein-protein interaction studies predicted the higher affinity of SpUlp1 towards SUMO compared to its counterpart from Saccharomyces cerevisiae (ScUlp1). The catalytic domain of SpUlp1 was purified using Ni-NTA chromatography with 83.33% recovery yield. Moreover, In vitro activity data further confirmed the fast-acting nature of SpUlp1 catalytic domain, where a 90% cleavage of fusion proteins was obtained within 1 h of incubation, indicating novelty and commercial relevance of S. pombe Ulp1. Biophysical characterization showed 8.8% α-helices, 36.7% β-sheets in SpUlp1SD. From thermal CD and fluorescence data, SpUlp1SD T m was found to be 45 °C. Further, bioprocess optimization using fed-batch cultivation resulted in 3.5 g/L of SpUlp1SD production with Y P/X of 77.26 mg/g DCW and volumetric productivity of 205.88 mg/L/h., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Human soluble guanylate cyclase (sGC) is a heme-containing metalloprotein in NO-sGC-cGMP signaling. In this work, fluorescent proteins were employed to study the NO-induced sGC molecular mechanism via mutagenesis at the catalytic domain. The conformational change of sGC by mutant α 1 C595 was investigated in living cells through fluorescence lifetime imaging microscopy (FLIM). The results indicated that the NO-induced conformational change of the catalytic domain of sGC from "open to "closed" upon GTP-binding was regulated by the hydrogen (H)-bonding network of the catalytic domain. The mutation of C595 caused a big conformational change of catalytic domain with H-bond variation, which not only demonstrates the key role of the C595 site in the process of conformational change of the catalytic domain, but also reveals the regulatory mechanism of sGC at the catalytic domain. This finding would guide the design of small-molecule drugs targeting the catalytic domain to modulate sGC activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

A mutant of ubiquitin C-terminal hydrolase L1 (UCHL1) detected in early-onset neurodegenerative patients, UCHL1 R178Q , showed higher catalytic activity than wild-type UCHL1 (UCHL1 WT ). Lying within the active-site pocket, the arginine is part of an interaction network that holds the catalytic histidine in an inactive arrangement. However, the structural basis and mechanism of enzymatic activation upon glutamine substitution was not understood. We combined X-ray crystallography, protein nuclear magnetic resonance (NMR) analysis, enzyme kinetics, covalent inhibition analysis, and biophysical measurements to delineate activating factors in the mutant. While the crystal structure of UCHL1 R178Q showed nearly the same arrangement of the catalytic residues and active-site pocket, the mutation caused extensive alteration in the chemical environment and dynamics of more than 30 residues, some as far as 15 Å away from the site of mutation. Significant broadening of backbone amide resonances in the HSQC spectra indicates considerable backbone dynamics changes in several residues, in agreement with solution small-angle X-ray scattering (SAXS) analyses which indicate an overall increase in protein flexibility. Enzyme kinetics show the activation is due to a k cat effect despite a slightly weakened substrate affinity. In line with this, the mutant shows a higher second-order rate constant (k inact /K i ) in a reaction with a substrate-derived irreversible inhibitor, Ub-VME, compared to the wild-type enzyme, an observation indicative of a more reactive catalytic cysteine in the mutant. Together, the observations underscore structural plasticity as a factor contributing to enzyme kinetic behavior which can be modulated through mutational effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Soluble guanylyl cyclase (GC-1) is the primary receptor of nitric oxide (NO) in smooth muscle cells and maintains vascular function by inducing vasorelaxation in nearby blood vessels. GC-1 converts guanosine 5'-triphosphate (GTP) into cyclic guanosine 3',5'-monophosphate (cGMP), which acts as a second messenger to improve blood flow. While much work has been done to characterize this pathway, we lack a mechanistic understanding of how NO binding to the heme domain leads to a large increase in activity at the C-terminal catalytic domain. Recent structural evidence and activity measurements from multiple groups have revealed a low-activity cyclase domain that requires additional GC-1 domains to promote a catalytically-competent conformation. How the catalytic domain structurally transitions into the active conformation requires further characterization. This review focuses on structure/function studies of the GC-1 catalytic domain and recent advances various groups have made in understanding how catalytic activity is regulated including small molecules interactions, Cys-S-NO modifications and potential interactions with the NO-sensor domain and other proteins., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Tankyrases (TNKS and TNKS2) are enzymes that catalyze poly-ADP-ribosylation (PARsylation) of their target proteins. Tankyrase-mediated PARsylation plays critical regulatory roles in cell signaling, particularly in the Wnt/β-catenin pathway. The sterile alpha motif (SAM) domain in tankyrases mediates their oligomerization, which is essential for tankyrase function. The oligomerization regulates the catalytic activity of tankyrases, but the underlying mechanism is unclear. Our analyses of crystal structures of the tankyrase catalytic domain suggest that formation of a head-to-head dimer regulates the catalytic activity. Our activity assays show that residues in the catalytic domain dimer interface are important for the PARsylation activity of tankyrases both in solution and cells. The dimer is weak and may only form in the context of the SAM domain-mediated oligomers of tankyrases, consistent with the dependence of the tankyrase activity on the SAM domain., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Previous studies of the protein kinase, ERK2, using NMR and hydrogen-exchange measurements have shown changes in dynamics accompanying its activation by phosphorylation. However, knowledge about the conformational motions involved is incomplete. Here, we examined ERK2 using long conventional molecular dynamics (MD) simulations starting from crystal structures of phosphorylated (2P) and unphosphorylated (0P) forms. Individual trajectories were run for (5 to 25) μs, totaling 727 μs. The results show unexpected flexibility of the A-loop, with multiple long-lived (>5 μs) conformational states in both 2P- and 0P-ERK2. Differential contact network and principal component analyses reveal coupling between the A-loop fold and active site dynamics, with evidence for conformational selection in the kinase core of 2P-ERK2 but not 0P-ERK2. Simulations of 2P-ERK2 show A-loop states corresponding to restrained dynamics within the N-lobe, including regions around catalytic residues. One A-loop conformer forms lasting interactions with the L16 segment, leading to reduced RMSF and greater compaction in the active site. By contrast, simulations of 0P-ERK2 reveal excursions of A-loop residues away from the C-lobe, leading to greater active site mobility. Thus, the A-loop in ERK2 switches between distinct conformations that reflect coupling with the active site, possibly via the L16 segment. Crystal packing interactions suggest that lattice contacts with the A-loop may restrain its structural variation in X-ray structures of ERK2. The novel conformational states identified by MD expand our understanding of ERK2 regulation, by linking the activated state of the kinase to reduced dynamics and greater compaction surrounding the catalytic site., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)