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The Campylobacter jejuni bacterium, which causes foodborne enteritis in humans, expresses the uncharacterized protein CJ0600. Based on sequence analysis, CJ0600 has been proposed to function as a 1-aminocyclopropane-1-carboxylate (ACC) deaminase (AccDA) or cysteine desulfhydrase (CysDS). However, it has never been investigated whether CJ0600 exerts AccDA or CysDS activity or how CJ0600 mediates its enzymatic activity. To reveal the structural features necessary for the function of CJ0600, we determined the crystal structure of CJ0600 and characterized its enzymatic activity. CJ0600 contains two domains and features an interdomain pocket, which accommodates a pyridoxal 5'-phosphate (PLP) molecule as a Schiff base with its lysine residue (K35), as observed in its structural homologs, including AccDA, CysDS, and serine deaminase (SerDA). However, unlike its structural homologs, CJ0600 exists as a monomer and exhibits unique structural features throughout its structure. Moreover, CJ0600 contains unique active site residues that are not observed in AccDA, CysDS, or SerDA. Consistently, phylogenetic analysis indicates that CJ0600 and its orthologs are evolutionarily distinct from AccDA, CysDS, and SerDA. Indeed, CJ0600 showed no CysDS or SerDA activity and extremely weak AccDA activity. These observations suggest that CJ0600 functions as a unique PLP-dependent enzyme that has not been reported., 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.)

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.)

Lactate dehydrogenase A (LDHA) is a key enzyme in Warburg's effect, a characteristic of cancer cells. LDHA is a target of anticancer agents that inhibit the metabolism of cancer cells. Gossypol is a known cancer therapeutic agent that inhibits LDHA by competitive inhibition. However, the mechanisms of inhibition of LDHA by gossypol is unknown. Here, we elucidate the binding of gossypol and LDHA using biochemical and biophysical methods. The crystal structure of the complex between LDHA and gossypol is presented. The binding of gossypol affects LDHA activity by a conformational change in the active-site loop. Our research contributes to the structural insight into LDHA with gossypol and approaches gossypol as a novel therapeutic candidate targeting the metabolic pathways for cancer cells., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

ß-Amylase, which catalyses the release of ß-anomeric maltose from the non-reducing end of starch, is widely used in the food industry. Increasing its enzyme activity through protein engineering might improve the efficiency of food processing. To obtain detailed structural information to assist rationale design, here the crystal structure of Bacillus cereus β-amylase (BCB) complexed with maltose was determined by molecular replacement and refined using anisotropic temperature factors to 1.26 Å resolution with R work /R free factors of 12.4/15.7 %. The structure contains six maltose and one glucose molecules, of which two maltose and one glucose are bound at sites not previously observed in BCB structures. These three new sugar-binding sites are located on the surface and likely to be important in enhancing the degradation of raw-starch granules. In the active site of BCB, two maltose molecules are bound in tandem at subsites -2 ∼ -1 and +1 ∼ +2. Notably, the conformation of the glucose moiety bound at subsite -1 is a mixture of α-anomeric distorted 1,4 B boat and 4 C 1 chair forms, while those at subsites -2, +1 ∼ +2 are all in the 4 C 1 chair forms. The O1 of the distorted α-glucose residue at subsite -1 occupies the position of the putative catalytic water, forming a hydrogen bond with OE1 of Glu367 (base catalyst), suggesting that this distorted sugar is not involved in catalysis. Together, these findings pave the way for further improving the functionality of microbial ß-amylase 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 Inc. All rights reserved.)

β-Galactosidase from Bacillus circulans ATCC 31382 (BgaD) is a biotechnologically important enzyme for the synthesis of β-galactooligosaccharides (GOS). Among its four isoforms, isoform A (BgaD-A) has distinct synthetic properties. Here, we present cryoelectron microscopy (cryo-EM) structures of BgaD-A and compare them with the known X-ray crystal structure of isoform D (BgaD-D), revealing substantial structural divergences between the two isoforms. In contrast to BgaD-D, BgaD-A features a flexible Big-4 domain and another enigmatic domain. The newly identified flexible region in BgaD-A is termed as "barrier domain 8," and serves as a barricade, obstructing the access of longer oligosaccharide substrates into the active site of BgaD-A. The transgalactosylation reactions catalyzed by both isoforms revealed that BgaD-A has a higher selectivity than BgaD-D in the earlier stages of the reaction and is prevailingly directed to shorter galactooligosaccharides. This study improves our understanding of the structural determinants governing β-galactosidase catalysis, with implications for tailored GOS production., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Enzymes of the 2-oxoacid:ferredoxin oxidoreductase (OFOR) superfamily catalyze the reversible oxidation of 2-oxoacids to acyl-coenzyme A esters and carbon dioxide (CO 2 )using ferredoxin or flavodoxin as the redox partner. Although members of the family share primary sequence identity, a variety of domain and subunit arrangements are known. Here, we characterize the structure of a four-subunit family member: the pyruvate:ferredoxin oxidoreductase (PFOR) from the methane producing archaeon Methanosarcina acetivorans (MaPFOR). The 1.92 Å resolution crystal structure of MaPFOR shows a protein fold like those of single- or two-subunit PFORs that function in 2-oxoacid oxidation, including the location of the requisite thiamine pyrophosphate (TPP), and three [4Fe-4S] clusters. Of note, MaPFOR typically functions in the CO 2 reductive direction, and structural comparisons to the pyruvate oxidizing PFORs show subtle differences in several regions of catalytical relevance. These studies provide a framework that may shed light on the biochemical mechanisms used to facilitate reductive pyruvate synthesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Non-heme iron-dependent sulfoxide/selenoxide synthases (NHISS) constitute a unique metalloenzyme class capable of installing a C-S/Se bond onto histidine to generate thio/selenoimidazole antioxidants, such as ergothioneine and ovothiol. These natural products are increasingly recognized for their health benefits. Among associated ergothioneine-biosynthetic enzymes, type IV EgtBs stand out, as they exhibit low sequence similarity with other EgtB subfamilies due to their recent divergence from the ovothiol-biosynthetic enzyme OvoA. Herein, we present crystal structures of two representative EgtB-IV enzymes, offering insights into the basis for this evolutionary convergence and enhancing our understanding of NHISS active site organization more broadly. The ability to interpret how key residues modulate substrate specificity and regioselectivity has implications for downstream identification of divergent reactivity within the NHISS family. To this end, we identify a previously unclassified clade of OvoA-like enzymes with a seemingly hybrid set of characteristics, suggesting they may represent an evolutionary intermediate between OvoA and EgtB-IV., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Lacto-N-neotetraose (LNnT), a representative oligosaccharide found in human milk, has been previously examined for its beneficial traits. However, the LNnT titer is limited by the efficient glycosyltransferase pathway, particularly with respect to the catalysis of rate-limiting steps. As data on the crystal structure of the key enzyme required for synthesizing LNnT are lacking, the synthesis of LNnT remains an uncertainty. Here, for the first time we report the three-dimensional structure of a bacterial β-1,4-galactosyltransferase, Aaβ4GalT, and analyze the critical role played by residues in its catalytic efficacy. Guided by structural insights, we engineered this enzyme to enhance its catalytic efficiency using structure-guided tunnel engineering. The mutant enzyme L5 (K155M/H156D/F157W/K185M/Q216V) so produced, showed a 50-fold enhancement in catalytic activity. Crystal structure analysis revealed that the mechanism underlying the improvement in activity was of the swing door type. The closed conformation formed by dense hydrophobic packing with Q216V-K155M widened and permitted substrate entry. Our results show that altering the tunnel conformation helped appropriately accommodate the substrate for catalysis and provide a structural basis for the modification of other glycosyltransferases., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To get a deeper understanding of the structural bases of the allosteric transition between T and R states of plant and bacterial phosphoenolpyruvate carboxylases (PEPCs), we obtained the first T-state crystal structures of the maize photosynthetic PEPC (ZmPEPC-C 4 ) and exhaustively compared them with the previously reported R-state ZmPEPC-C 4 and other T-state structures. We identified previously unrecognized significant conformational changes in the T state: that of the α8-α9 loop, which connects the two kinds of activator allosteric sites with the active site, the conversion of the α30 helix into a 3 10 helix, leading to the disorganization of the active site lid and activators allosteric sites, and the closure of the inhibitor allosteric-site lid. Additionally, we identified previously overlooked, highly conserved residues of potential interest in the allosteric transition, including two histidines whose protonation might stabilize the T state. The crystal structures reported here also suggest similar tetrameric quaternary arrangements of PEPC enzymes in the R and T states, and the location of the bicarbonate binding site, as well as the conformational changes required for the carboxylation step. Our findings and working hypothesis advance the understanding of the structural features of the allosteric PEPC enzymes and provide a foundation for future experiments., Competing Interests: Declaration of competing interest The authors declare no conflict of interes., (Copyright © 2024. Published by Elsevier B.V.)

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) have a major role in the physiology of eukaryotic cells by mediating reactive oxygen species production. Evolutionarily distant proteins with the NOX catalytic core have been found in bacteria, including Streptococcus pneumoniae NOX (SpNOX), which is proposed as a model for studying NOXs because of its high activity and stability in detergent micelles. We present here cryo-electron microscopy structures of substrate-free and nicotinamide adenine dinucleotide (NADH)-bound SpNOX and of NADPH-bound wild-type and F397A SpNOX under turnover conditions. These high-resolution structures provide insights into the electron-transfer pathway and reveal a hydride-transfer mechanism regulated by the displacement of F397. We conducted structure-guided mutagenesis and biochemical analyses that explain the absence of substrate specificity toward NADPH and suggest the mechanism behind constitutive activity. Our study presents the structural basis underlying SpNOX enzymatic activity and sheds light on its potential in vivo function., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Argonaute (Ago) proteins are programmable nucleases found in all domains of life, playing a crucial role in biological processes like DNA/RNA interference and gene regulation. Mesophilic prokaryotic Agos (pAgos) have gained increasing research interest due to their broad range of potential applications, yet their molecular mechanisms remain poorly understood. Here, we present seven cryo-electron microscopy structures of Kurthia massiliensis Ago (KmAgo) in various states. These structures encompass the steps of apo-form, guide binding, target recognition, cleavage, and release, revealing that KmAgo employs a unique DDD catalytic triad, instead of a DEDD tetrad, for DNA target cleavage under 5'P-DNA guide conditions. Notably, the last catalytic residue, D713, is positioned outside the catalytic pocket in the absence of guide. After guide binding, D713 enters the catalytic pocket. In contrast, the corresponding catalytic residue in other Agos has been consistently located in the catalytic pocket. Moreover, we identified several sites exhibiting enhanced catalytic activity through alanine mutagenesis. These sites have the potential to serve as engineering targets for augmenting the catalytic efficiency of KmAgo. This structural analysis of KmAgo advances the understanding of the diversity of molecular mechanisms by Agos, offering insights for developing and optimizing mesophilic pAgos-based programmable DNA and RNA manipulation tools., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Protein glycation is a universal, non-enzymatic modification that occurs when a sugar covalently attaches to a primary amine. These spontaneous modifications may have deleterious or regulatory effects on protein function, and their removal is mediated by the conserved metabolic kinase fructosamine-3-kinase (FN3K). Despite its crucial role in protein repair, we currently have a poor understanding of how FN3K engages or phosphorylates its substrates. By integrating structural biology and biochemistry, we elucidated the catalytic mechanism for FN3K-mediated protein deglycation. Our work identifies key amino acids required for binding and phosphorylating glycated substrates and reveals the molecular basis of an evolutionarily conserved protein repair pathway. Additional structural-functional studies revealed unique structural features of human FN3K as well as differences in the dimerization behavior and regulation of FN3K family members. Our findings improve our understanding of the structure of FN3K and its catalytic mechanism, which opens new avenues for therapeutically targeting FN3K., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Aging workers of the termite Neocapritermes taracua can defend their colony by sacrificing themselves by body rupture, mixing the externally stored blue laccase BP76 with hydroquinones to produce a sticky liquid rich in toxic benzoquinones. Here, we describe the crystal structure of BP76 isolated from N. taracua in its native form. The structure reveals several stabilization strategies, including compact folding, glycosylation, and flexible loops with disulfide bridges and tight dimer interface. The remarkable stability of BP76 maintains its catalytic activity in solid state during the lifespan of N. taracua workers, providing old workers with an efficient defensive weapon to protect their colony., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Guanosine 5'-monophosphate (GMP) synthetase (GuaA) catalyzes the last step of GMP synthesis in the purine nucleotide biosynthetic pathway. This enzyme catalyzes a reaction in which xanthine 5'-monophosphate (XMP) is converted to GMP in the presence of Gln and ATP through an adenyl-XMP intermediate. A structure of an XMP-bound form of GuaA from the domain Bacteria has not yet been determined. In this study, the crystal structure of an XMP-bound form of GuaA from the thermophilic bacterium Thermus thermophilus HB8 (TtGuaA) was determined at a resolution of 2.20 Å and that of an apo form of TtGuaA was determined at 2.10 Å resolution. TtGuaA forms a homodimer, and the monomer is composed of three domains, which is a typical structure for GuaA. Disordered regions in the crystal structure were obtained from the AlphaFold2-predicted model structure, and a model with substrates (Gln, XMP and ATP) was constructed for molecular-dynamics (MD) simulations. The structural fluctuations of the TtGuaA dimer as well as the interactions between the active-site residues were analyzed by MD simulations., (open access.)

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.)

Substituted tetrahydrofuran derivatives were designed and synthesized to serve as the P2 ligand for a series of potent HIV-1 protease inhibitors. Both enantiomers of the tetrahydrofuran derivatives were synthesized stereoselectivity in optically active forms using lipase-PS catalyzed enzymatic resolution as the key step. These tetrahydrofuran derivatives are designed to promote hydrogen bonding and van der Waals interactions with the backbone atoms in the S2 subsite of the HIV-1 protease active site. Several inhibitors displayed very potent HIV-1 protease inhibitory activity. A high-resolution X-ray crystal structure of an inhibitor-bound HIV-1 protease provided important insight into the ligand binding site interactions in the active site.

Bacteroides fragilis resides in mammals and human intestines and secrete series of proteins and molecules outside that cause various diseases such as colon cancer and chronic colitis in the host. B. fragilis has been shown to produce numerous proteins to the infected cell surface which are involved in host colonization, microbial interactions, and pathogenicity. Among secreted proteins, a B. fragilis toxin (BFT) is a metalloprotease and disintegrates the epithelial cell layer and causes colon cancers. Except the BFT, information of secreted proteases from B. fragilis is limited and no structure is available. Aspartic proteinase cleaves a peptide bond using two aspartate residues in a catalytic site in acidic conditions, pH ranges from 3 to 6. Aspartic proteinase have been characterized mostly from eukaryotes and retroviruses but rare from bacteria including B. fragilis. A putative aspartic proteinase is identified from the B. fragilis genome and prepared recombinantly as a Bacteroides aspartic proteinase (BAPtase). The crystal structure of BAPtase was determined at 2.6 Å. Structure-based comparative and endopeptidase analyses demonstrated that BAPtase presents a two-domain structure and is a functional aspartic proteinase in unusually weak basic pHs, which would propose to be a critical in bacterial pathogenesis and in host immunity. Our observations on the distinct structural and catalytic properties of BAPtase would benefit the future development of B. fragilis-specific drugs or preventatives., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The BisI family of restriction endonucleases is unique in requiring multiple methylated or hydroxymethylated cytosine residues within a short recognition sequence (GCNGC), and in cleaving directly within this sequence, rather than at a distance. Here, we report that the number of modified cytosines that are required for cleavage can be tuned by the salt concentration. We present crystal structures of two members of the BisI family, NhoI and Eco15I_Ntd (N-terminal domain of Eco15I), in the absence of DNA and in specific complexes with tetra-methylated GCNGC target DNA. The structures show that NhoI and Eco15I_Ntd sense modified cytosine bases in the context of double-stranded DNA (dsDNA) without base flipping. In the co-crystal structures of NhoI and Eco15I_Ntd with DNA, the internal methyl groups (G5mCNGC) interact with the side chains of an (H/R)(V/I/T/M) di-amino acid motif near the C-terminus of the distal enzyme subunit and arginine residue from the proximal subunit. The external methyl groups (GCNG5mC) interact with the proximal enzyme subunit, mostly through main chain contacts. Surface plasmon resonance analysis for Eco15I_Ntd shows that the internal and external methyl binding pockets contribute about equally to sensing of cytosine methyl groups., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In this report, we structurally and biochemically characterized the unknown gene product SP1746 from Streptococcus pneumoniae serotype 4. Various crystal structures of SP1746 in the apo form and in complex with different nucleotides were determined. SP1746 is a globular protein, which belongs to the histidine-aspartate (HD) domain superfamily with two Fe 3+ ions in the active site that are coordinated by key active site residues and water molecules. All nucleotides bind in a similar orientation in the active site with their phosphate groups anchored to the diiron cluster. Biochemically, SP1746 hydrolyzes different nucleotide substrates. SP1746 most effectively hydrolyzes diadenosine tetraphosphate (Ap4A) to two ADPs. Based on the aforementioned data, we annotated SP1746 as an Ap4A hydrolase, belonging to the YqeK family. Our in vitro data indicate a potential role for SP1746 in regulating Ap4A homeostasis, which requires validation with in vivo experiments in bacteria in the future., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The emergence of drug-resistant bacteria, facilitated by metallo-beta-lactamases (MBLs), presents a significant obstacle to the effective use of antibiotics in the management of clinical drug-resistant bacterial infections. AFM-1 is a MBL derived from Alcaligenes faecalis and shares 86% homology with the NDM-1 family. Both AFM-1 and NDM-1 demonstrate the ability to hydrolyze ampicillin and other β-lactam antibiotics, however, their substrate affinities vary, and the specific reason for this variation remains unknown. We present the high-resolution structure of AFM-1. The active center of AFM-1 binds two zinc ions, and the conformation of the key amino acid residues in the active center is in accordance with that of NDM-1. However, the substrate-binding pocket of AFM-1 is considerably smaller than that of NDM-1. Additionally, the mutation of amino acid residues in the Loop3 region, as compared to NDM-1, results in the formation of a dense hydrophobic patch comprised of hydrophobic amino acid residues in this area, which facilitates substrate binding. Our findings lay the foundation for understanding the molecular mechanism of AFM-1 with a high affinity for substrates and provide a novel theoretical foundation for addressing the issue of drug resistance caused by B1 MBLs., 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.)

Glycoside hydrolase (GH) family 13 is among the main families of enzymes acting on starch; recently, subfamily 47 of GH13 (GH13_47) has been established. The crystal structure and function of a GH13_47 enzyme from Bacteroides ovatus has only been reported to date. This enzyme has α-amylase activity, while the GH13_47 enzymes comprise approximately 800-900 amino acid residues which are almost double those of typical α-amylases. It is important to know how different the GH13_47 enzymes are from other α-amylases. Rhodothermus marinus JCM9785, a thermophilic bacterium, possesses a gene for the GH13_47 enzyme, which is designated here as RmGH13_47A. Its structure has been predicted to be composed of seven domains: N1, N2, N3, A, B, C, and D. We constructed a plasmid encoding Gly266-Glu886, which contains the N3, A, B, and C domains and expressed the protein in Escherichia coli. The enzyme hydrolyzed starch and pullulan by a neopullulanase-type action. Additionally, the enzyme acted on maltotetraose, and saccharides with α-1,6-glucosidic linkages were observed in the products. Following the replacement of the catalytic residue Asp563 with Ala, the crystal structure of the variant D563A in complex with the enzymatic products from maltotetraose was determined; as a result, electron density for an α-1,6-branched pentasaccharide was observed in the catalytic pocket, and Ile762 and Asp763 interacted with the branched chain of the pentasaccharide. These findings suggest that RmGH13_47A is an α-amylase that prefers α-1,6-branched parts of starch to produce oligosaccharides., (© 2024 Wiley Periodicals LLC.)

Enteroviruses cause a wide range of disorders with varying presentations and severities, and some enteroviruses have emerged as serious public health concerns. These include Coxsackievirus B3 (CVB3), an active causative agent of viral myocarditis, and Coxsackievirus B4 (CVB4), which may accelerate the progression of type 1 diabetes. The 3C proteases from CVB3 and CVB4 play important roles in the propagation of these viruses. In this study, the 3C proteases from CVB3 and CVB4 were expressed in Escherichia coli and purified by affinity chromatography and gel-filtration chromatography. The crystals of the CVB3 and CVB4 3C proteases diffracted to 2.10 and 2.01 Å resolution, respectively. The crystal structures were solved by the molecular-replacement method and contained a typical chymotrypsin-like fold and a conserved His40-Glu71-Cys147 catalytic triad. Comparison with the structures of 3C proteases from other enteroviruses revealed high similarity with minor differences, which will guide the design of 3C-targeting inhibitors with broad-spectrum properties.

The superfamily of acid proteases has two catalytic aspartates for proteolysis of their peptide substrates. Here, we show a minimal structural scaffold, the structural catalytic core (SCC), which is conserved within each family of acid proteases, but varies between families, and thus can serve as a structural marker of four individual protease families. The SCC is a dimer of several structural blocks, such as the DD-link, D-loop, and G-loop, around two catalytic aspartates in each protease subunit or an individual chain. A dimer made of two (D-loop + DD-link) structural elements makes a DD-zone, and the D-loop + G-loop combination makes a psi-loop. These structural markers are useful for protein comparison, structure identification, protein family separation, and protein engineering.

Human flavin adenine dinucleotide synthase (hFADS) is a bifunctional, multi-domain enzyme that exhibits both flavin mononucleotide adenylyltransferase and pyrophosphatase activities. Here we report the crystal structure of full-length hFADS2 and its C-terminal PAPS domain in complex with flavin adenine dinucleotide (FAD), and dissect the structural determinants underlying the contribution of each individual domain, within isoforms 1 and 2, to each of the two enzymatic activities. Structural and functional characterization performed on complete or truncated constructs confirmed that the C-terminal domain tightly binds FAD and catalyzes its synthesis, while the combination of the N-terminal molybdopterin-binding and KH domains is the minimal essential substructure required for the hydrolysis of FAD and other ADP-containing dinucleotides. hFADS2 associates in a stable C2-symmetric dimer, in which the packing of the KH domain of one protomer against the N-terminal domain of the other creates the adenosine-specific active site responsible for the hydrolytic activity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pseudoalteromonas fuliginea sp. PS47 is a recently identified marine bacterium that has extensive enzymatic machinery to metabolize polysaccharides, including a locus that targets pectin-like substrates. This locus contains a gene (locus tag EU509_03255) that encodes a pectin-degrading lyase, called PfPL1, that belongs to polysaccharide lyase family 1 (PL1). The 2.2 Å resolution X-ray crystal structure of PfPL1 reveals the compact parallel β-helix fold of the PL1 family. The back side of the core parallel β-helix opposite to the active site is a meandering set of five α-helices joined by lengthy loops. A comparison of the active site with those of other PL1 enzymes suggests a catalytic mechanism that is independent of metal ions, such as Ca 2+ , but that substrate recognition may require metal ions. Overall, this work provides the first structural insight into a pectinase of marine origin and the first structure of a PL1 enzyme in subfamily 2., (open access.)

Endolysosomal exonucleases PLD3 and PLD4 (phospholipases D3 and D4) are associated with autoinflammatory and autoimmune diseases. We report structures of these enzymes, and the molecular basis of their catalysis. The structures reveal an intra-chain dimer topology forming a basic active site at the interface. Like other PLD superfamily members, PLD3 and PLD4 carry HxKxxxxD/E motifs and participate in phosphodiester-bond cleavage. The enzymes digest ssDNA and ssRNA in a 5'-to-3' manner and are blocked by 5'-phosphorylation. We captured structures in apo, intermediate, and product states and revealed a "link-and-release" two-step catalysis. We also unexpectedly demonstrated phosphatase activity via a covalent 3-phosphohistidine intermediate. PLD4 contains an extra hydrophobic clamp that stabilizes substrate and could affect oligonucleotide substrate preference and product release. Biochemical and structural analysis of disease-associated mutants of PLD3/4 demonstrated reduced enzyme activity or thermostability and the possible basis for disease association. Furthermore, these findings provide insight into therapeutic design., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Base excision repair (BER) is a critical genome defense pathway that copes with a broad range of DNA lesions induced by endogenous or exogenous genotoxic agents. AP endonucleases in the BER pathway are responsible for removing the damaged bases and nicking the abasic sites. In plants, the BER pathway plays a critical role in the active demethylation of 5-methylcytosine (5mC) DNA modification. Here, we have determined the crystal structures of Arabidopsis AP endonuclease AtARP in complex with the double-stranded DNA containing tetrahydrofuran (THF) that mimics the abasic site. We identified the critical residues in AtARP for binding and removing the abasic site and the unique residues for interacting with the orphan base. Additionally, we investigated the differences among the three plant AP endonucleases and evaluated the general DNA repair capacity of AtARP in a mammalian cell line. Our studies provide further mechanistic insights into the BER pathway in plants., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Crystal structures of human long-chain acyl-CoA dehydrogenase (LCAD) and the catalytically inactive Glu291Gln mutant, have been determined. These structures suggest that LCAD harbors functions beyond its historically defined role in mitochondrial β-oxidation of long and medium-chain fatty acids. LCAD is a homotetramer containing one FAD per 43 kDa subunit with Glu291 as the catalytic base. The substrate binding cavity of LCAD reveals key differences which makes it specific for longer and branched chain substrates. The presence of Pro132 near the start of the E helix leads to helix unwinding that, together with adjacent smaller residues, permits binding of bulky substrates such as 3α, 7α, l2α-trihydroxy-5β-cholestan-26-oyl-CoA. This structural element is also utilized by ACAD11, a eucaryotic ACAD of unknown function, as well as bacterial ACADs known to metabolize sterol substrates. Sequence comparison suggests that ACAD10, another ACAD of unknown function, may also share this substrate specificity. These results suggest that LCAD, ACAD10, ACAD11 constitute a distinct class of eucaryotic acyl CoA dehydrogenases., (© 2024. The Author(s).)

Itaconyl-CoA hydratase in Pseudomonas aeruginosa (PaIch) converts itaconyl-CoA to (S)-citramalyl-CoA upon addition of a water molecule, a part of an itaconate catabolic pathway in virulent organisms required for their survival in humans host cells. Crystal structure analysis of PaIch showed that a unique N-terminal hotdog fold containing a 4-residue short helical segment α3-, named as an "eaten sausage", followed by a flexible loop region slipped away from the conserved β-sheet scaffold, whereas the C-terminal hotdog fold is similar to all MaoC. A conserved hydratase motif with catalytic residues provides mechanistic insights into catalysis, and existence of a longer substrate binding tunnel may suggest the binding of longer CoA derivatives., (© 2024 Federation of European Biochemical Societies.)

The biosynthesis pathways of coenzyme A (CoA) in most archaea involve several unique enzymes including dephospho-CoA kinase (DPCK) that converts dephospho-CoA to CoA in the final step of CoA biosynthesis in all domains of life. The archaeal DPCK is unrelated to the analogous bacterial and eukaryotic enzymes and shows no significant sequence similarity to any proteins with known structures. Unusually, the archaeal DPCK utilizes GTP as the phosphate donor although the analogous bacterial and eukaryotic enzymes are ATP-dependent kinases. Here, we report the crystal structure of DPCK and its complex with GTP and a magnesium ion from the archaeal hyperthermophile Thermococcus kodakarensis. The crystal structure demonstrates why GTP is the preferred substrate of this kinase. We also report the activity analyses of site-directed mutants of crucial residues determined based on sequence conservation and the crystal structure. From these results, the key residues involved in the reaction of phosphoryl transfer and the possible dephospho-CoA binding site are inferred., (© 2024 Wiley Periodicals LLC.)

Apoptosis-inducing factor (AIF), which is confined to mitochondria of normal healthy cells, is the first identified caspase-independent cell death effector. Moreover, AIF is required for the optimal functioning of the respiratory chain machinery. Recent findings have revealed that AIF fulfills its pro-survival function by interacting with CHCHD4, a soluble mitochondrial protein which promotes the entrance and the oxidative folding of different proteins in the inner membrane space. Here, we report the crystal structure of the ternary complex involving the N-terminal 27-mer peptide of CHCHD4, NAD + , and AIF harboring its FAD (flavin adenine dinucleotide) prosthetic group in oxidized form. Combining this information with biophysical and biochemical data on the CHCHD4/AIF complex, we provide a detailed structural description of the interaction between the two proteins, validated by both chemical cross-linking mass spectrometry analysis and site-directed mutagenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of Xenopus laevis polα-primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5' end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer-template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα-primase., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

3-mercaptopropionate (3MPA) dioxygenase (MDO) is a mononuclear nonheme iron enzyme that catalyzes the O 2 -dependent oxidation of thiol-bearing substrates to yield the corresponding sulfinic acid. MDO is a member of the cysteine dioxygenase family of small molecule thiol dioxygenases and thus shares a conserved sequence of active site residues (Serine-155, Histidine-157, and Tyrosine-159), collectively referred to as the SHY-motif. It has been demonstrated that these amino acids directly interact with the mononuclear Fe-site, influencing steady-state catalysis, catalytic efficiency, O 2 -binding, and substrate coordination. However, the underlying mechanism by which this is accomplished is poorly understood. Here, pulsed electron paramagnetic resonance spectroscopy [ 1 H Mims electron nuclear double resonance spectroscopy] is applied to validate density functional theory computational models for the MDO Fe-site simultaneously coordinated by substrate and nitric oxide (NO), (3MPA/NO)-MDO. The enhanced resolution provided by electron nuclear double resonance spectroscopy allows for direct observation of Fe-bound substrate conformations and H-bond donation from Tyr159 to the Fe-bound NO ligand. Further inclusion of SHY-motif residues within the validated model reveals a distinct channel restricting movement of the Fe-bound NO-ligand. It has been argued that the iron-nitrosyl emulates the structure of potential Fe(III)-superoxide intermediates within the MDO catalytic cycle. While the merit of this assumption remains unconfirmed, the model reported here offers a framework to evaluate oxygen binding at the substrate-bound Fe-site and possible reaction mechanisms. It also underscores the significance of hydrogen bonding interactions within the enzymatic active site., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Phototransduction in retinal rods occurs when the G protein-coupled photoreceptor rhodopsin triggers the activation of phosphodiesterase 6 (PDE6) by GTP-bound alpha subunits of the G protein transducin (Gα T ). Recently, we presented a cryo-EM structure for a complex between two GTP-bound recombinant Gα T subunits and native PDE6, that included a bivalent antibody bound to the C-terminal ends of Gα T and the inhibitor vardenafil occupying the active sites on the PDEα and PDEβ subunits. We proposed Gα T -activated PDE6 by inducing a striking reorientation of the PDEγ subunits away from the catalytic sites. However, questions remained including whether in the absence of the antibody Gα T binds to PDE6 in a similar manner as observed when the antibody is present, does Gα T activate PDE6 by enabling the substrate cGMP to access the catalytic sites, and how does the lipid membrane enhance PDE6 activation? Here, we demonstrate that 2:1 Gα T -PDE6 complexes form with either recombinant or retinal Gα T in the absence of the Gα T antibody. We show that Gα T binding is not necessary for cGMP nor competitive inhibitors to access the active sites; instead, occupancy of the substrate binding sites enables Gα T to bind and reposition the PDE6γ subunits to promote catalytic activity. Moreover, we demonstrate by reconstituting Gα T -stimulated PDE6 activity in lipid bilayer nanodiscs that the membrane-induced enhancement results from an increase in the apparent binding affinity of Gα T for PDE6. These findings provide new insights into how the retinal G protein stimulates rapid catalytic turnover by PDE6 required for dim light vision., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A considerable number of lytic polysaccharide monooxygenases (LPMOs) and other carbohydrate-active enzymes are modular, with catalytic domains being tethered to additional domains, such as carbohydrate-binding modules, by flexible linkers. While such linkers may affect the structure, function, and stability of the enzyme, their roles remain largely enigmatic, as do the reasons for natural variation in length and sequence. Here, we have explored linker functionality using the two-domain cellulose-active ScLPMO10C from Streptomyces coelicolor as a model system. In addition to investigating the WT enzyme, we engineered three linker variants to address the impact of both length and sequence and characterized these using small-angle X-ray scattering, NMR, molecular dynamics simulations, and functional assays. The resulting data revealed that, in the case of ScLPMO10C, linker length is the main determinant of linker conformation and enzyme performance. Both the WT and a serine-rich variant, which have the same linker length, demonstrated better performance compared with those with either a shorter linker or a longer linker. A highlight of our findings was the substantial thermostability observed in the serine-rich variant. Importantly, the linker affects thermal unfolding behavior and enzyme stability. In particular, unfolding studies show that the two domains unfold independently when mixed, whereas the full-length enzyme shows one cooperative unfolding transition, meaning that the impact of linkers in biomass-processing enzymes is more complex than mere structural tethering., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The bacterial cell wall consists of a three-dimensional peptidoglycan layer, composed of peptides linked to the sugars N-acetylmuramic acid (MurNAc) and GlcNAc. Unlike other bacteria, the pathogenic Tannerella forsythia, a member of the red complex group of bacteria associated with the late stages of periodontitis, lacks biosynthetic pathways for MurNAc production and therefore obtains MurNAc from the environment. Sugar kinases play a crucial role in the MurNAc recycling process, activating the sugar molecules by phosphorylation. In this study, we present the first crystal structures of a MurNAc kinase, called murein sugar kinase (MurK), in its unbound state as well as in complexes with the ATP analog β-γ-methylene adenosine triphosphate (AMP-PCP) and with MurNAc. We also determined the crystal structures of K1058, a paralogous MurNAc kinase of T. forsythia, in its unbound state and in complex with MurNAc. We identified the active site and residues crucial for MurNAc specificity as the less bulky side chains of S133, P134, and L135, which enlarge the binding cavity for the lactyl ether group, unlike the glutamate or histidine residues present in structural homologs. In establishing the apparent kinetic parameters for both enzymes, we showed a comparable affinity for MurNAc (K m 180 μM and 30 μM for MurK and K1058, respectively), with MurK being over two hundred times faster than K1058 (V max 80 and 0.34 μmol min -1  mg -1 , respectively). These data might support a structure-guided approach to development of inhibitory MurNAc analogs for pathogen MurK enzymes., Competing Interests: Conflicts of interest The authors declare that they no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

G-protein metallochaperones are essential for the proper maturation of numerous metalloenzymes. The G-protein chaperone MMAA in humans (MeaB in bacteria) uses GTP hydrolysis to facilitate the delivery of adenosylcobalamin (AdoCbl) to AdoCbl-dependent methylmalonyl-CoA mutase, an essential metabolic enzyme. This G-protein chaperone also facilitates the removal of damaged cobalamin (Cbl) for repair. Although most chaperones are standalone proteins, isobutyryl-CoA mutase fused (IcmF) has a G-protein domain covalently attached to its target mutase. We previously showed that dimeric MeaB undergoes a 180° rotation to reach a state capable of GTP hydrolysis (an active G-protein state), in which so-called switch III residues of one protomer contact the G-nucleotide of the other protomer. However, it was unclear whether other G-protein chaperones also adopted this conformation. Here, we show that the G-protein domain in a fused system forms a similar active conformation, requiring IcmF oligomerization. IcmF oligomerizes both upon Cbl damage and in the presence of the nonhydrolyzable GTP analog, guanosine-5'-[(β,γ)-methyleno]triphosphate, forming supramolecular complexes observable by mass photometry and EM. Cryo-EM structural analysis reveals that the second protomer of the G-protein intermolecular dimer props open the mutase active site using residues of switch III as a wedge, allowing for AdoCbl insertion or damaged Cbl removal. With the series of structural snapshots now available, we now describe here the molecular basis of G-protein-assisted AdoCbl-dependent mutase maturation, explaining how GTP binding prepares a mutase for cofactor delivery and how GTP hydrolysis allows the mutase to capture the cofactor., Competing Interests: Conflict of interest C. L. D. is a Howard Hughes Medical Investigator. M. J. consults for Gate Biosciences and Evozyne. The other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Human Histone Deacetylase 2 (HDAC2) belongs to a conserved enzyme superfamily that regulates deacetylation inside cells. HDAC2 is a drug target as it is known to be upregulated in cancers and neurodegenerative disorders. It consists of globular deacetylase and C-terminus intrinsically-disordered domains [1-3]. To date, there is no full-length structure of HDAC2 available due to the high intrinsic flexibility of its C-terminal domain. The intrinsically-disordered domain, however, is known to be important for the enzymatic function of HDAC2 [1, 4]. Here we combine several structural Mass Spectrometry (MS) methodologies such as denaturing, native, ion mobility and chemical crosslinking, alongside biochemical assays and molecular modelling to study the structure and dynamics of the full-length HDAC2 for the first time. We show that MS can easily dissect heterogeneity inherent within the protein sample and at the same time probe the structural arrangement of the different conformers present. Activity assays combined with data from MS and molecular modelling suggest how the structural dynamics of the C-terminal domain, and its interactions with the catalytic domain, regulate the activity of this enzyme., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Rift Valley fever virus (RVFV) belongs to the order Bunyavirales and is the type species of genus Phlebovirus , which accounts for over 50% of family Phenuiviridae species. RVFV is mosquito-borne and causes severe diseases in both humans and livestock, and consists of three segments (S, M, L) in the genome. The L segment encodes an RNA-dependent RNA polymerase (RdRp, L protein) that is responsible for facilitating the replication and transcription of the virus. It is essential for the virus and has multiple drug targets. Here, we established an expression system and purification procedures for full-length L protein, which is composed of an endonuclease domain, RdRp domain, and cap-binding domain. A cryo-EM L protein structure was reported at 3.6 Å resolution. In this first L protein structure of genus Phlebovirus , the priming loop of RVFV L protein is distinctly different from those of other L proteins and undergoes large movements related to its replication role. Structural and biochemical analyses indicate that a single template can induce initiation of RNA synthesis, which is notably enhanced by 5' viral RNA. These findings help advance our understanding of the mechanism of RNA synthesis and provide an important basis for developing antiviral inhibitors. IMPORTANCE The zoonosis RVF virus (RVFV) is one of the most serious arbovirus threats to both human and animal health. RNA-dependent RNA polymerase (RdRp) is a multifunctional enzyme catalyzing genome replication as well as viral transcription, so the RdRp is essential for studying the virus and has multiple drug targets. In our study, we report the structure of RVFV L protein at 3.6 Å resolution by cryo-EM. This is the first L protein structure of genus Phlebovirus . Strikingly, a single template can initiate RNA replication. The structure and assays provide a comprehensive and in-depth understanding of the catalytic and substrate recognition mechanism of RdRp.

Pseudouridine (Ψ) is a ubiquitous RNA modification incorporated by pseudouridine synthase (Pus) enzymes into hundreds of noncoding and protein-coding RNA substrates. Here, we determined the contributions of substrate structure and protein sequence to binding and catalysis by pseudouridine synthase 7 (Pus7), one of the principal messenger RNA (mRNA) modifying enzymes. Pus7 is distinct among the eukaryotic Pus proteins because it modifies a wider variety of substrates and shares limited homology with other Pus family members. We solved the crystal structure of Saccharomyces cerevisiae Pus7, detailing the architecture of the eukaryotic-specific insertions thought to be responsible for the expanded substrate scope of Pus7. Additionally, we identified an insertion domain in the protein that fine-tunes Pus7 activity both in vitro and in cells. These data demonstrate that Pus7 preferentially binds substrates possessing the previously identified UG U AR (R = purine) consensus sequence and that RNA secondary structure is not a strong requirement for Pus7-binding. In contrast, the rate constants and extent of Ψ incorporation are more influenced by RNA structure, with Pus7 modifying UG U AR sequences in less-structured contexts more efficiently both in vitro and in cells. Although less-structured substrates were preferred, Pus7 fully modified every transfer RNA, mRNA, and nonnatural RNA containing the consensus recognition sequence that we tested. Our findings suggest that Pus7 is a promiscuous enzyme and lead us to propose that factors beyond inherent enzyme properties (e.g., enzyme localization, RNA structure, and competition with other RNA-binding proteins) largely dictate Pus7 substrate selection., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Amine transaminases (ATAs) are pyridoxal-5'-phosphate (PLP)-dependent enzymes that catalyze the transfer of an amino group from an amino donor to an aldehyde and/or ketone. In the past decade, the enzymatic reductive amination of prochiral ketones catalyzed by ATAs has attracted the attention of researchers, and more traditional chemical routes were replaced by enzymatic ones in industrial manufacturing. In the present work, the influence of the presence of an α,β-unsaturated system in a methylketone model substrate was investigated, using a set of five wild-type ATAs, the ( R )-selective from Aspergillus terreus (Atr-TA) and Mycobacterium vanbaalenii (Mva-TA), the ( S )-selective from Chromobacterium violaceum (Cvi-TA), Ruegeria pomeroyi (Rpo-TA), V. fluvialis (Vfl-TA) and an engineered variant of V. fluvialis (ATA-256 from Codexis). The high conversion rate (80 to 99%) and optical purity (78 to 99% ee ) of both ( R )- and ( S )-ATAs for the substrate 1-phenyl-3-butanone, using isopropylamine (IPA) as an amino donor, were observed. However, the double bond in the α,β-position of 4-phenylbut-3-en-2-one dramatically reduced wild-type ATA reactivity, leading to conversions of <10% (without affecting the enantioselectivity). In contrast, the commercially engineered V. fluvialis variant, ATA-256, still enabled an 87% conversion, yielding a corresponding amine with >99% ee . Computational docking simulations showed the differences in orientation and intermolecular interactions in the active sites, providing insights to rationalize the observed experimental results.

Caffeic acid O-methyltransferase from Ligusticum chuanxiong (LcCOMT) showed strict regiospecificity despite a relative degree of preference. Compared with caffeic acid, methyl caffeate was the preferential substrate by its low Km and high Kcat. In this study, we obtained the SAM binary (1.80 Å) and SAH binary (1.95 Å) complex LcCOMT crystal structures, and established the ternary complex structure with methyl caffeate by molecular docking. The active site of LcCOMT included phenolic substrate pocket, SAM/SAH ligand pocket and conserved catalytic residues as well. The regiospecificity of LcCOMT that permitted only 3-hydroxyl group to be methylated arise from the interactions between the active site and the phenyl ring. However, the propanoid tail governed the relative preference of LcCOMT. The ester group in methyl caffeate stabilized the anionic intermediate caused by His268-Asp269 pair, whereas caffeic acid was unable to stabilize the anionic intermediate due to the adjacent carboxylate anion in the propanoid tail. Ser183 residue formed an additional hydrogen bond with SAH and its role was identified by S183A mutation. Ile318 residue might be a potential site for determination of substrate preference, and its mutation led to the change of tertiary conformation. The results supported the selective mechanism of LcCOMT., (Copyright © 2021. Published by Elsevier B.V.)

Understanding the selectivity mechanism of inhibitors towards homology proteins helps to design selective candidates. Phosphodiesterase (PDE) family members act in the degradation of cAMP and cGMP, among which some isoforms such as PDE9A are attracting interest for Alzheimer's disease treatment, while PDE10A is used as target for treating schizophrenia. In this study, computational methods were used to investigate the major features of PDE9A/10A, with the purpose to provide deep understanding of the molecular mechanism of selective inhibition towards these two isoforms. Our result revealed that two conserved residues Gln453 and Phe456 were proven to be crucial for the binding affinity and inhibitory selectivity of PDE9A inhibitors. In addition, the high-affinity PDE9A inhibitors always interact with the conservative hydrophobic pocket as well as Tyr424 and Ala452 of PDE9A, while PDE10A selective inhibitors need to have two hydrophobic groups and two hydrogen bond donors to interact with the conservative Tyr693, Gln726, and Phe729 of PDE10A. This study provides valuable insights into the underlying mechanism of selective inhibition targeting PDE9A and PDE10A, for further search for potent and highly selective PDE9A/10A inhibitors., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5' exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3'-to-5' ExoN domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14's enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

During HIV-1 infection, the envelope glycoprotein subunit gp41 folds into a six-helix bundle structure (6HB) formed by the interaction between its N-terminal (NHR) and C-terminal (CHR) heptad-repeats, promoting viral and cell membranes fusion. A highly preserved, hydrophobic pocket (HP) on the NHR surface is crucial in 6HB formation and, therefore, HP-binding compounds constitute promising therapeutics against HIV-1. Here, we investigated the conformational and dynamic properties of the HP using a rationally designed single-chain protein (named covNHR) that mimics the gp41 NHR structure. We found that the fluorescent dye 8-anilino-naphtalene-1-sulfonic acid (ANS) binds specifically to the HP, suggesting that ANS derivatives may constitute lead compounds to inhibit 6HB formation. ANS shows different binding modes to the HP, depending on the occupancy of other NHR pockets. Moreover, in presence of a CHR peptide bound to the N-terminal pockets in gp41, two ANS molecules can occupy the HP showing cooperative behavior. This binding mode was assessed using molecular docking and molecular dynamics simulations. The results show that the HP is conformationally flexible and connected allosterically to other NHR regions, which strongly influence the binding of potential ligands. These findings could guide the development of small-molecule HIV-1 inhibitors targeting the HP., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Nucleoside 2'-deoxyribosyltransferases (NDTs) catalyze the cleavage of glycosidic bonds of 2'-deoxynucleosides and the following transfer of the 2'-deoxyribose moiety to acceptor nucleobases. Here, we report the crystal structures and biochemical properties of the first tetrameric NDTs: the type I NDT from the mesophilic bacterium Enterococcus faecalis V583 (EfPDT) and the type II NDT from the bacterium Desulfotalea psychrophila (DpNDT), the first psychrophilic NDT. This novel structural and biochemical data permitted an exhaustive comparative analysis aimed to shed light into the basis of the high global stability of the psychrophilic DpNDT, which has a higher melting temperature than EfPDT (58.5 °C versus 54.4 °C) or other mesophilic NDTs. DpNDT possesses a combination of unusual structural motifs not present neither in EfPDT nor any other NDT that most probably contribute to its global stability, in particular, a large aliphatic isoleucine-leucine-valine (ILV) bundle accompanied by a vicinal disulfide bridge and also an intersubunit disulfide bridge, the first described for an NDT. The functional and structural features of DpNDT do not fit the standard features of psychrophilic enzymes, which lead us to consider the implication of (sub)cellular levels together with the protein level in the adaptation of enzymatic activity to low temperatures., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Although myriad protein-protein interactions in nature use polyvalent binding, in which multiple ligands on one entity bind to multiple receptors on another, to date an affinity advantage of polyvalent binding has been demonstrated experimentally only in cases where the target receptor molecules are clustered prior to complex formation. Here, we demonstrate cooperativity in binding affinity (i.e., avidity) for a protein complex in which an engineered dimer of the amyloid precursor protein inhibitor (APPI), possessing two fully functional inhibitory loops, interacts with mesotrypsin, a soluble monomeric protein that does not self-associate or cluster spontaneously. We found that each inhibitory loop of the purified APPI homodimer was over three-fold more potent than the corresponding loop in the monovalent APPI inhibitor. This observation is consistent with a suggested mechanism whereby the two APPI loops in the homodimer simultaneously and reversibly bind two corresponding mesotrypsin monomers to mediate mesotrypsin dimerization. We propose a simple model for such dimerization that quantitatively explains the observed cooperativity in binding affinity. Binding cooperativity in this system reveals that the valency of ligands may affect avidity in protein-protein interactions including those of targets that are not surface-anchored and do not self-associate spontaneously. In this scenario, avidity may be explained by the enhanced concentration of ligand binding sites in proximity to the monomeric target, which may favor rebinding of the multiple ligand binding sites with the receptor molecules upon dissociation of the protein complex., Competing Interests: The authors have declared that no competing interests exist.

Jatropha curcas contains seeds with a high oil content, suitable for biodiesel production. After oil extraction, the remaining mass can be a rich source of enzymes. However, data from the literature describing physicochemical characteristics for a monomeric esterase from the J. curcas seed did not fit the electrostatic catapult model for esterases/lipases. We decided to reevaluate this J. curcas esterase and extend its characterization to check this apparent discrepancy and gain insights into the enzyme's potential as a biocatalyst. After anion exchange chromatography and two-dimensional gel electrophoresis, we identified the enzyme as belonging to the dienelactone hydrolase family, characterized by a cysteine as the nucleophile in the catalytic triad. The enzyme displayed a basic optimum hydrolysis pH of 9.0 and an acidic pI range, in contrast to literature data, making it well in line with the electrostatic catapult model. Furthermore, the enzyme showed low hydrolysis activity in an organic solvent-containing medium (isopropanol, acetonitrile, and ethanol), which reverted when recovering in an aqueous reaction mixture. This enzyme can be a valuable tool for hydrolysis reactions of short-chain esters, useful for pharmaceutical intermediates synthesis, due to both its high hydrolytic rate in basic pH and its stability in an organic solvent.

Fucoidans are sulfated, fucose-rich marine polysaccharides primarily found in cell walls of brown seaweeds (macroalgae). Fucoidans are known to possess beneficial bioactivities depending on their structure and sulfation degree. Here, we report the first functional characterization and the first crystal structure of a prokaryotic sulfatase, PsFucS1, belonging to sulfatase subfamily S1&#95;13, able to release sulfate from fucoidan oligosaccharides. PsFucS1 was identified in the genome of a Pseudoalteromonas sp. isolated from sea cucumber gut. PsFucS1 (57 kDa) is Ca 2+ dependent and has an unusually high optimal temperature (68 °C) and thermostability. Further, the PsFucS1 displays a unique quaternary hexameric structure comprising a tight trimeric dimer complex. The structural data imply that this hexamer formation results from an uncommon interaction of each PsFucS1 monomer that is oriented perpendicular to the common dimer interface (~ 1500 Å 2 ) that can be found in analogous sulfatases. The uncommon interaction involves interfacing (1246 Å 2 ) through a bundle of α-helices in the N-terminal domain to form a trimeric ring structure. The high thermostability may be related to this unusual quaternary hexameric structure formation that is suggested to represent a novel protein thermostabilization mechanism., (© 2021. The Author(s).)