Search

Streptomyces griseolus CYP105A1 exhibits monooxygenase activity to a wide variety of structurally different substrates with regio- and stereospecificity, making its application range broad. Our previous studies have shown that CYP105A1 wild type and its variants metabolize 12 types of nonsteroidal anti-inflammatory drugs (NSAIDs). In particular, the R84A variant exhibited a high activity against many NSAIDs. We successfully crystallized complexes of wild-type CYP105A1 (WT) and the R84A variant with diclofenac (DIF) or flufenamic acid (FLF). In the WT, the carboxyl group of DIF formed a charged hydrogen bond with Arg84. In contrast, in R84A, the carboxyl group formed two bidentate charged hydrogen bonds with Arg73. The C4' atom of the benzene ring of DIF, which undergoes hydroxylation by WT and R84A, was positioned approximately 4 Å from the heme iron. Binding of FLF was nearly the same in both WT and R84A. The carboxyl group of FLF formed charged hydrogen bonds with Arg73. In both WT and R84A, FLF appeared to be fixed by this charged hydrogen bonding with Arg73 during the reaction, and the C4' atom, which undergoes hydroxylation, must face the heme iron. Thus, the dihedral angles of the two N-C bonds connecting the two benzene rings of FLF needed to rotate by 78° and -71°, respectively. The temperature factors of the F-G loop, helix F, and helix G of R84A were remarkably higher than those of WT. This suggests that these regions in R84A are much more flexible compared to those of WT, which may consequently affect substrate binding and product release.

Agmatine, a natural polyamine also known as 4-aminobutyl-guanidine, is biosynthesized from arginine by decarboxylation. Aspergillus oryzae contains high amounts of agmatine, suggesting highly active arginine decarboxylase (ADC) in this organism. However, genome analysis revealed no ADC homolog in A. oryzae. A. oryzae strain RIB40 has six homologs of phosphatidylserine decarboxylase (PSD), an enzyme that synthesizes phosphatidyl ethanolamine from phosphatidylserine. We previously discovered that one of these homologs, AO090102000327, encodes arginine decarboxylase, which we named ADC1. In the present study, we determined the crystal structures of ligand-free, arginine-treated, and agmatine-treated ADC1 each at 1.9-2.15 Å resolution. Each structure contained four ADC1 molecules (chains A-D) in the asymmetric unit of the cell. Each ADC1 molecule is a heterodimer consisting of the N-terminal region (Asn60-Gly441) and C-terminal region (Ser442-Thr482). In the ligand-free ADC1, the N-terminus of Ser442 was modified to form a pyruvoyl group. In the arginine-treated ADC1, arginine was converted to agmatine, with the pyruvoyl group covalently bound to agmatine by forming a Schiff base. The same structure was observed in agmatine-treated ADC1. These results indicate that ADC1 is a pyruvoyl-dependent decarboxylase and unveils the reaction mechanism of ADC from A. oryzae., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interestsKiyoshi Yasukawa reports financial support was provided by Japan Society for the Promotion of Science. 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 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.)

In utero colonization or deposition of beneficial microorganisms and their by-products likely occurs through various mechanisms, such as hematogenous spread or ascension from the reproductive tract. With high-throughput sequencing techniques, the identification of microbial components in first-pass neonatal meconium has been achieved. While these components are low-biomass and often not abundant enough to culture, the presence of microbial DNA signatures may promote fetal immune tolerance or epigenetic regulation prior to birth. The aim of this study was to investigate the maternal source of the neonatal first-pass meconium microbiome. Maternal vaginal and anal samples collected from twenty-one maternal-infant dyad pairs were compared via principal component analysis to first-pass neonatal meconium microbial compositions. Results demonstrated the greatest degree of similarity between the maternal gut microbiome in the second and third trimesters and vaginal microbiome samples across pregnancy, suggesting that the maternal gut microbiota may translocate to the fetal gut during pregnancy. This study sheds light on the origin and timing of the potential transfer of maternal microbial species to offspring in utero.

The bacterial Efe system functions as an importer of free Fe 2+ into cells independently of iron-chelating compounds such as siderophores and consisted of iron-binding protein EfeO, peroxidase EfeB, and transmembrane permease EfeU. While we and other researchers reported crystal structures of EfeO and EfeB, that of EfeU remains undetermined. In this study, we constructed expression system of EfeU derived from Escherichia coli, selected E. coli Rosetta-gami 2 (DE3) as an expression host, and succeeded in purification of the proteins which were indicated to form an oligomer by blue native PAGE. We obtained preliminary data of the X-ray crystallography, suggesting that expression and purification methods we established in this study enable structural analysis of the bacterial Efe system., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest associated with this manuscript., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Thaumatin is a sweet-tasting protein that elicits a sweet taste at a threshold of approximately 50 nM. Structure-sweetness relationships in thaumatin suggest that the basicity of two amino acids residues, Arg82 and Lys67, are particularly responsible for sweetness. Using tetragonal crystals, our structural analysis suggested that flexible sidechain conformations of these two residues play an important role in sweetness. However, in tetragonal crystals, Arg82 is adjacent to symmetry-related residues, and its flexibility is relatively restrained by the crystal packing. To reduce and diminish these symmetry-related effects, orthorhombic crystals were prepared, and their structures were successfully determined at a resolution of 0.89 Å. Within the orthorhombic lattice, two alternative conformations were more clearly visible at Lys67 than in a tetragonal system. Interestingly, for the first time, three alternative conformations at Arg82 were only found in an orthorhombic system. These results suggest the importance of flexible conformations in sweetness determinants. Such subtle structural variations might serve to adjust the complementarity of the electrostatic potentials of sweet receptors, thereby eliciting the potent sweet taste of thaumatin., 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.)

XynR is a thermostable alkaline GH10 xylanase, for which we have previously examined the effects of saturation mutagenesis at position 315 on enzyme alkaliphily, and found that at pH 10, the activities of variants could be ordered as follows: T315Q > T315S = T315N > T315H = wild-type XynR (WT) > 15 other variants. In this study, we sought to elucidate the mechanisms underlying the variable activity of these different variants. Crystallographic analysis revealed that the Ca 2+ ion near position 315 in WT was absent in the T315Q variant. We accordingly hypothesized that the enhancement of alkaliphily in T315Q, and probably also in the T315H, T315N, and T315S variants, could be ascribed to an activity-stability trade-off associated with a reduction in stability due to the lack of this Ca 2+ ion. Consistent with expectations, the alkaline resistance of T315H, T315N, T315Q, and T315S, evaluated through the pH-dependence of stability at 0 mM CaCl 2 under alkaline conditions, was found to be lower than that of WT: the residual activity at pH 11 of WT was 78% while those of T315H, T315N, T315Q, and T315S were 0, 9, 0, and 43%, respectively. In addition, the thermostabilities of these four variants, as assessed using the denaturing temperatures (T m ) at 0 mM CaCl 2 based on ellipticity at 222 nm in circular dichroism measurements, were lower than that of WT by 2-8 °C. Furthermore, the T m values of WT and variants at 5 mM CaCl 2 were higher than those at 0 mM CaCl 2 by 6-11 °C. Collectively, our findings in this study indicate that mutation of the T residue at position 315 of XynR to H, N, Q, and S causes an increase in the alkaliphily of this enzyme, thereby reducing its stability., (© 2024. The Author(s).)

Lignin, flavonoids, melatonin, and stilbenes are plant specialized metabolites with diverse physiological and biological functions, supporting plant growth and conferring stress resistance. Their biosynthesis requires O-methylations catalyzed by 5-hydroxyconiferaldehyde O-methyltransferase (CAldOMT; also called caffeic acid O-methyltransferase, COMT). CAldOMT was first known for its roles in syringyl (S) lignin biosynthesis in angiosperm cell walls and later found to be multifunctional. This enzyme also catalyzes O-methylations in flavonoid, melatonin, and stilbene biosynthetic pathways. Phylogenetic analysis indicated the convergent evolution of enzymes with OMT activities towards the monolignol biosynthetic pathway intermediates in some gymnosperm species that lack S-lignin and Selaginella moellendorffii, a lycophyte which produces S-lignin. Furthermore, neofunctionalization of CAldOMTs occurred repeatedly during evolution, generating unique O-methyltransferases (OMTs) with novel catalytic activities and/or accepting novel substrates, including lignans, 1,2,3-trihydroxybenzene, and phenylpropenes. This review summarizes multiple aspects of CAldOMTs and their related proteins in plant metabolism and discusses their evolution, molecular mechanism, and roles in biorefineries, agriculture, and synthetic biology., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

mRNA export is an essential pathway for the regulation of gene expression. In humans, closely related RNA helicases, UAP56 and URH49, shape selective mRNA export pathways through the formation of distinct complexes, known as apo-TREX and apo-AREX complexes, and their subsequent remodeling into similar ATP-bound complexes. Therefore, defining the unidentified components of the apo-AREX complex and elucidating the molecular mechanisms underlying the formation of distinct apo-complexes is key to understanding their functional divergence. In this study, we identify additional apo-AREX components physically and functionally associated with URH49. Furthermore, by comparing the structures of UAP56 and URH49 and performing an integrated analysis of their chimeric mutants, we exhibit unique structural features that would contribute to the formation of their respective complexes. This study provides insights into the specific structural and functional diversification of these two helicases that diverged from the common ancestral gene Sub2., (© 2024. The Author(s).)

The depolymerization of lignocellulosic biomass is facilitated by feruloyl esterases (FAEs), which hydrolyze ester bonds between lignin and polysaccharides. Fungal FAEs belonging to subfamily (SF) 6 release precursors such as ferulic acid derivatives, attractive for biochemical production. Among these, Aspergillus sydowii FAE (AsFaeE), an SF6 FAE, exhibits remarkable activity across various substrates. In this study, we conducted X-ray crystallography and kinetic analysis to unravel the molecular mechanisms governing substrate recognition and catalysis by AsFaeE. AsFaeE exhibits a typical α/β-hydrolase fold, characterized by a catalytic triad of serine, aspartate, and histidine. Comparative analysis of substrate-free, ferulic acid-bound, and sinapic acid-bound forms of AsFaeE suggests a conformational change in the loop covering the substrate-binding pocket upon binding. Notably, Pro158 and Phe159 within this loop cover the phenolic part of the substrate, forming three layers of planar rings. Our structure-based functional mutagenesis clarifies the roles of the residues involved in substrate binding and catalytic activity. Furthermore, distinct substrate-binding mechanisms between AsFaeE and other studied FAEs are identified. This investigation offers the initial structural insights into substrate recognition by SF6 FAEs, equipping us with structural knowledge that might facilitate the design of FAE variants capable of efficiently processing a wider range of substrate sizes., 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. Published by Elsevier B.V.)

Tetrahydrofuran ring formation from dibenzylbutyrolactone lignans is a key step in the biosynthesis of aryltetralin lignans including deoxypodophyllotoxin and podophyllotoxin. Previously, Fe(II)- and 2-oxoglutarate-dependent dioxygenase (2-ODD) from Podophyllum hexandrum (Himalayan mayapple, Berberidaceae) was found to catalyze the cyclization of a dibenzylbutyrolactone lignan, yatein, to give deoxypodophyllotoxin and designated as deoxypodophyllotoxin synthase (DPS). Recently, we reported that the biosynthesis of deoxypodophyllotoxin and podophyllotoxin evolved in a lineage-specific manner in phylogenetically unrelated plant species such as P. hexandrum and Anthriscus sylvestris (cow parsley, Apiaceae). Therefore, a comprehensive understanding of the characteristics of DPSs that catalyze the cyclization of yatein to deoxypodophyllotoxin in various plant species is important. However, for plant species other than P. hexandrum, the isolation of the DPS enzyme gene and the type of the enzyme, e.g. whether it is 2-ODD or another type of enzyme such as cytochrome P-450, have not been reported. In this study, we report the identification and characterization of A. sylvestris DPS (AsDPS). Phylogenetic analysis showed that AsDPS belonged to the 2-ODD superfamily and shared moderate amino acid sequence identity (40.8%) with P. hexandrum deoxypodophyllotoxin synthase (PhDPS). Recombinant protein assay indicated that AsDPS and PhDPS differ in terms of the selectivity of substrate enantiomers. Protein modeling using AlphaFold2 and site-directed mutagenesis indicated that the Tyr305 residue of AsDPS probably contributes to substrate recognition. This study advances our understanding of the podophyllotoxin biosynthetic pathway in A. sylvestris and provides new insight into 2-ODD involved in plant secondary (specialized) metabolism., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Some probiotics including lactobacilli, colonize host animal cells by targeting glycosaminoglycans (GAGs), such as heparin, located in the extracellular matrix. Recent studies have shown that several lactic acid bacteria degrade GAGs. Here we show the structure/function relationship of Lacticaseibacillus rhamnosus 4-deoxy-L- threo -5-hexosulose-uronate ketol-isomerase (KduI) crucial for metabolism of unsaturated glucuronic acid produced through degradation of GAGs. Crystal structures of ligand-free and bound KduIs were determined by X-ray crystallography and the enzyme was found to consist of six identical subunits and adopt a β-helix as a basic scaffold. Ligands structurally similar to the substrate were bound to the cleft of each enzyme subunit. Several residues located in the cleft interacted with ligands through hydrogen bonds and/or C-C contacts. In addition to substrate analogs, a metal ion coordinated to four residues, His198, His200, Glu205, and His248, in the cleft, and the enzyme activity was significantly inhibited by a chelator, ethylenediaminetetraacetic acid. Site-directed mutants in Arg163, Ile165, Thr184, Thr194, His200, Arg203, Tyr207, Met262, and Tyr269 in the cleft exhibited little enzyme activity, indicating that these residues and the metal ion constituted an active site in the cleft. This is the first report on the active site structure of KduI based on the ligand-bound complex., Competing Interests: The authors declare no conflict of interests., (2023 by The Japanese Society of Applied Glycoscience.)

Grimontia hollisae collagenase (Ghcol) exhibits high collagen-degrading activity. To explore its catalytic mechanism, its substrate (Gly-Pro-Hyp-Gly-Pro-Hyp, GPOGPO)-complexed crystal structure was determined at 2.0 Å resolution. A water molecule was observed near the active-site zinc ion. Since this water was not observed in the product (GPO)-complexed Ghcol, it was hypothesized that the GPOGPO-complexed Ghcol structure reflects a Michaelis complex, providing a structural basis for understanding the catalytic mechanism. Analyses of the active-site geometry and site-directed mutagenesis of the active-site tyrosine residues revealed that Glu493 and Tyr564 were essential for catalysis, suggesting that Glu493 functions as an acid and base catalyst while Tyr564 stabilizes the tetrahedral complex in the transition state. These results shed light on the catalytic mechanism of bacterial collagenase., (© 2023 Federation of European Biochemical Societies.)

Thaumatin is an intensely sweet-tasting protein. Its sweetness persists when heated under acidic conditions, but disappears when heated at a pH above 7.0. To clarify how the structural features of thaumatin resist insoluble aggregation during heating under acidic conditions, we analysed its crystal structure obtained at pH 4.0, 6.0, and 8.0. Simultaneously, the melting temperature (Tm) at these pH levels was determined using differential scanning fluorimetry. At pH 4.0, the Tm of thaumatin was substantially lower and the overall B-factor value of its structure was higher than those at pH 6.0. Interestingly, the relative B-factor values for most lysine residues decreased as the pH reduced. These results suggest that the overall structure at pH 4.0 becomes flexible but the relative flexibility of some regions is lower than that at pH 6.0. Thus, the reduction in relative flexibility might play an important role in preventing thermal aggregation, thereby maintaining the sweetness., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Collagenase from the gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently even than clostridial collagenase, the most widely used industrial collagenase. However, the structural determinants facilitating this efficiency are unclear. Here, we report the crystal structures of ligand-free and Gly-Pro-hydroxyproline (Hyp)-complexed Ghcol at 2.2 and 2.4 Å resolution, respectively. These structures revealed that the activator and peptidase domains in Ghcol form a saddle-shaped structure with one zinc ion and four calcium ions. In addition, the activator domain comprises two homologous subdomains, whereas zinc-bound water was observed in the ligand-free Ghcol. In the ligand-complexed Ghcol, we found two Gly-Pro-Hyp molecules, each bind at the active site and at two surfaces on the duplicate subdomains of the activator domain facing the active site, and the nucleophilic water is replaced by the carboxyl oxygen of Hyp at the P1 position. Furthermore, all Gly-Pro-Hyp molecules bound to Ghcol have almost the same conformation as Pro-Pro-Gly motif in model collagen (Pro-Pro-Gly) 10 , suggesting these three sites contribute to the unwinding of the collagen triple helix. A comparison of activities revealed that Ghcol exhibits broader substrate specificity than clostridial collagenase at the P2 and P2' positions, which may be attributed to the larger space available for substrate binding at the S2 and S2' sites in Ghcol. Analysis of variants of three active-site Tyr residues revealed that mutation of Tyr564 affected catalysis, whereas mutation of Tyr476 or Tyr555 affected substrate recognition. These results provide insights into the substrate specificity and mechanism of G. hollisae collagenase., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Gram-negative Sphingomonas sp. strain A1 exhibits positive chemotaxis toward acidic polysaccharide pectin. SPH1118 has been identified as a pectin-binding protein involved in both pectin chemotaxis and assimilation. Here we show tertiary structures of SPH1118 with six different conformations as determined by X-ray crystallography. SPH1118 consisted of two domains with a large cleft between the domains and substrates bound to positively charged and aromatic residues in the cleft through hydrogen bond and stacking interactions. Substrate-free SPH1118 adopted three different conformations in the open form. On the other hand, the two domains were closed in substrate-bound form and the domain closure ratio was changed in response to the substrate size, suggesting that the conformational change upon binding to the substrate triggered the expression of pectin chemotaxis and assimilation. This study first clarified that the solute-binding protein with dual functions recognized the substrate through flexible conformational changes in response to the substrate size., (© 2022. The Author(s).)

Rhesus monkeys have evolved MHC-encoded class I allomorphs such as Mamu-B∗098 that are capable of binding N-myristoylated short lipopeptides rather than conventional long peptides; however, it remains unknown whether such antigen-binding molecules exist in other species, including humans. We herein demonstrate that human leukocyte antigen (HLA)-A∗24:02 and HLA-C∗14:02 proteins, which are known to bind conventional long peptides, also have the potential to bind N-myristoylated short lipopeptides. These HLA class I molecules shared a serine at position 9 (Ser9) with Mamu-B∗098, in contrast to most MHC class I molecules that harbor a larger amino acid residue, such as tyrosine, at this position. High resolution X-ray crystallographic analyses of lipopeptide-bound HLA-A∗24:02 and HLA-C∗14:02 complexes indicated that Ser9 was at the bottom of the B pocket with its small hydroxymethyl side chain directed away from the B-pocket cavity, thereby contributing to the formation of a deep hydrophobic cavity suitable for accommodating the long-chain fatty acid moiety of lipopeptide ligands. Upon peptide binding, however, we found the hydrogen-bond network involving Ser9 was reorganized, and the remodeled B pocket was able to capture the second amino acid residue (P2) of peptide ligands. Apart from the B pocket, virtually no marked alterations were observed for the A and F pockets upon peptide and lipopeptide binding. Thus, we concluded that the structural flexibility of the large B pocket of HLA-A∗2402 and HLA-C∗1402 primarily accounted for their previously unrecognized capacity to bind such chemically distinct ligands as conventional peptides and N-myristoylated lipopeptides., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest regarding the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

EfeUOB is a siderophore-independent iron uptake mechanism in bacteria. EfeU, EfeO, and EfeB are a permease, an iron-binding or electron-transfer protein, and a peroxidase, respectively. A Gram-negative bacterium, Sphingomonas sp. strain A1, encodes EfeU, EfeO, EfeB together with alginate-binding protein Algp7, a truncated EfeO-like protein (EfeO II ), in the genome. The typical EfeO (EfeO I ) consists of N-terminal cupredoxin and C-terminal M75 peptidase domains. Here, we detail the structure and function of bacterial EfeB and EfeO. Crystal structures of strain A1 EfeB and Escherichia coli EfeO I were determined at 2.30 Å and 1.85 Å resolutions, respectively. A molecule of heme involved in oxidase activity was bound to the C-terminal Dyp peroxidase domain of EfeB. Two domains of EfeO I were connected by a short loop, and a zinc ion was bound to four residues, Glu156, Glu159, Asp173, and Glu255, in the C-terminal M75 peptidase domain. These residues formed tetrahedron geometry suitable for metal binding and are well conserved among various EfeO proteins including Algp7 (EfeO II ), although the metal-binding site (HxxE) is proposed in the C-terminal M75 peptidase domain. This is the first report on structure of a typical EfeO with two domains, postulating a novel metal-binding motif "ExxE-//-D-//-E" in the EfeO C-terminal M75 peptidase domain., 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.)

Proper anther and pollen development are important for plant reproduction. The plant hormone gibberellin is important for anther development in rice, but its gametophytic functions remain largely unknown. Here, we report the functional and evolutionary analyses of rice gibberellin 3-oxidase 1 (OsGA3ox1), a gibberellin synthetic enzyme specifically expressed in the late developmental stages of anthers. Enzymatic and X-ray crystallography analyses reveal that OsGA3ox1 has a higher GA 7 synthesis ratio than OsGA3ox2. In addition, we generate an osga3ox1 knockout mutant by genome editing and demonstrate the bioactive gibberellic acid synthesis by the OsGA3ox1 action during starch accumulation in pollen via invertase regulation. Furthermore, we analyze the evolution of Oryza GA3ox1s and reveal that their enzyme activity and gene expression have evolved in a way that is characteristic of the Oryza genus and contribute to their male reproduction ability., (© 2022. The Author(s).)

Purpose: Extended reality (XR) technology enhances learning in medical education. The purpose of this study was to develop and apply a case-based approach for teaching radiological anatomy utilizing XR technology for improved student exploration and engagement., Methods: The workflow consisted of MRI scanning cadavers followed by radiological, pathological, and anatomical assessment, and finally case presentation based on XR visualizations and student interaction. Case information (Subject, History, and Physical Exam) was presented to student groups who generated and recorded hypotheses using Google Forms., Results: Use of all components of the system was voluntary and a total of 74 students responded to the survey request (response rate = 95%). Assessment of the experience was conducted through a qualitative survey comprising four Likert scale questions (1-5, 1 lowest), three binary questions, and open-ended comments. Mean, standard deviation, and overall agreement (mean ± SD, OA) showed that students found MRI scans of cadavers to be helpful for dissections (4.14 ± 1.1, 74.3%) and provided an understanding of relevant anatomy (4.32 ± 0.9, 79.7%), while 78.4% of students used the DICOM viewer to visualize scans of cadavers. The difficulty of use was found to be average (2.90 ± 1.0, 23%). zSpace visualizations were used by 40.5% of students, generally agreeing that an understanding of spatial relationships improved as a result (3.60 ± 1.0, 43.2%). More case-based sessions were favored by 97.3% of students., Conclusions: Results suggest that cadaveric MRI radiological visualization and XR technology enhance understanding of case-based anatomical dissections and encourage student exploration and engagement., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Gram-negative Sphingomonas sp. A1 incorporates acidic polysaccharide alginate into the cytoplasm via a cell-surface alginate-binding protein (AlgQ2)-dependent ATP-binding cassette transporter (AlgM1M2SS). We investigated the function of calcium bound to the EF-hand-like motif in AlgQ2 by introducing mutations at the calcium-binding site. The X-ray crystallography of the AlgQ2 mutant (D179A/E180A) demonstrated the absence of calcium binding and significant disorder of the EF-hand-like motif. Distinct from the wild-type AlgQ2, the mutant was quite unstable at temperature of strain A1 growth, although unsaturated alginate oligosaccharides stabilized the mutant by formation of substrate/protein complex. In the assay of ATPase and alginate transport by AlgM1M2SS reconstructed in the liposome, the wild-type and mutant AlgQ2 induced AlgM1M2SS ATPase activity in the presence of unsaturated alginate tetrasaccharide. These results indicate that the calcium bound to EF-hand-like motif stabilizes the substrate-unbound AlgQ2 but is not required for the complexation of substrate-bound AlgQ2 and AlgM1M2SS., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The mechanism of thermostabilization of GH10 xylanase, XynR, from Bacillus sp. strain TAR-1 by the mutation of S92 to E was investigated. Thermodynamic analysis revealed that thermostabilization was driven by the decrease in entropy change of activation for thermal inactivation. Crystallographic analysis suggested that this mutation suppressed the fluctuation of the amino acid residues at position 92-95., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The high-valent iron-oxo species formed in the non-heme diiron enzymes have high oxidative reactivity and catalyze difficult chemical reactions. Although the hydroxylation of inert methyl groups is an industrially promising reaction, utilizing non-heme diiron enzymes as such a biocatalyst has been difficult. Here we show a three-component monooxygenase system for the selective terminal hydroxylation of α-aminoisobutyric acid (Aib) into α-methyl-D-serine. It consists of the hydroxylase component, AibH1H2, and the electron transfer component. Aib hydroxylation is the initial step of Aib catabolism in Rhodococcus wratislaviensis C31-06, which has been fully elucidated through a proteome analysis. The crystal structure analysis revealed that AibH1H2 forms a heterotetramer of two amidohydrolase superfamily proteins, of which AibHm2 is a non-heme diiron protein and functions as a catalytic subunit. The Aib monooxygenase was demonstrated to be a promising biocatalyst that is suitable for bioprocesses in which the inert C-H bond in methyl groups need to be activated.

The covalent conjugation of a 14-carbon fatty acid (myristic acid) to the N-terminal Gly residue, termed N-myristoylation, occurs in some viral proteins to dictate their pathological function. This protein lipidation reaction, however, is monitored by host cytotoxic T lymphocytes that are capable of recognizing N-terminal lipopeptide fragments in the context of major histocompatibility complex (MHC) class I molecules. In a rhesus model of human AIDS, for example, the classical MHC class I allomorph, Mamu-B*05104, was shown to bind SIV Nef-derived 4-mer lipopeptides (myristic acid-Gly-Gly-Ala-Ile; C14nef4) and present them to the CD8+ T-cell line, SN45. These lipopeptides accommodated in MHC class I molecules expose much shorter peptide chains than conventional MHC class I-presented 8-10-mer peptides, and the molecular mechanisms by which αβ T-cell receptors (TCRs) recognize lipopeptides currently remain unclear. An X-ray crystallographic analysis of the SN45 TCR α and β heterodimer in a form that was co-crystallized with the C14nef4-bound Mamu-B*05104 complex indicated that the amide group of the N-myristoylated glycine residue offered a primary T-cell epitope by establishing a sole hydrogen bond between its nitrogen atom and the side chain of Glu at position 101 of CDR3β. Accordingly, the Glu to Ala mutation at this position resulted in the loss of lipopeptide recognition. On the other hand, TCRs were positioned remotely from the peptide portion of C14nef4, and strong interactions were not observed. Thus, these observations provide novel structural insights into lipopeptide recognition by TCRs, which contrast sharply with the general molecular principle of peptide recognition., (© The Japanese Society for Immunology. 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Saprophytic bacteria and plants compete for limited nutrient sources. Bacillus subtilis grows well on steamed soybeans Glycine max to produce the fermented food, natto. Here we focus on bacterial responses in conflict between B. subtilis and G. max. B. subtilis cells maintained high growth rates specifically on non-germinating, dead soybean seeds. On the other hand, viable soybean seeds with germinating capability attenuated the initial growth of B. subtilis. Thus, B. subtilis cells may trigger saprophytic growth in response to the physiological status of G. max. Scanning electron microscope observation indicated that B. subtilis cells on steamed soybeans undergo morphological changes to form apertures, demonstrating cell remodeling during saprophytic growth. Further, transcriptomic analysis of B. subtilis revealed upregulation of the gene cluster, yesOPQR, in colonies growing on steamed soybeans. Recombinant YesO protein, a putative, solute-binding protein for the ATP-binding cassette transporter system, exhibited an affinity for pectin-derived oligosaccharide from plant cell wall. The crystal structure of YesO, in complex with the pectin oligosaccharide, was determined at 1.58 Å resolution. This study expands our knowledge of defensive and offensive strategies in interspecies competition, which may be promising targets for crop protection and fermented food production.

Hemocyanin (Hc) and phenoloxidase (PO) are members of the type 3 copper protein family. Although arthropod Hc and PO exhibit similar three-dimensional structures of the copper-containing active site, Hc functions as an oxygen transport protein, showing minimal or no phenoloxidase activity. Here, we present the crystal structure of the oxy form of Hc from Panulirus japonicus (PjHc) at 1.58 Å resolution. The structure of the di-copper active site of PjHc was found to be almost identical to that of PO. Although conserved amino acids and the water molecule crucial for the enzymatic activity were observed in PjHc at almost the same positions as those in PO, PjHc showed no enzymatic activity under our experimental conditions. One striking difference between PjHc and arthropod PO was the presence of a "blocker residue" near the binuclear copper site of PjHc. This blocker residue comprised a phenylalanine residue tightly stacked with an imidazole ring of a CuA coordinated histidine and hindered substrates from accessing the active site. Our results suggest that the blocker residue is also a determining factor of the catalytic activity of type 3 copper proteins., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Newly synthesized major histocompatibility complex (MHC) class I proteins are stabilized in the endoplasmic reticulum (ER) by binding 8-10-mer-long self-peptide antigens that are provided by transporter associated with antigen processing (TAP). These MHC class I:peptide complexes then exit the ER and reach the plasma membrane, serving to sustain the steady-state MHC class I expression on the cell surface. A novel subset of MHC class I molecules that preferentially bind lipid-containing ligands rather than conventional peptides was recently identified. The primate classical MHC class I allomorphs, Mamu-B*098 and Mamu-B*05104, are capable of binding the N -myristoylated 5-mer (C14-Gly-Gly-Ala-Ile-Ser) or 4-mer (C14-Gly-Gly-Ala-Ile) lipopeptides derived from the N -myristoylated SIV Nef protein, respectively, and of activating lipopeptide antigen-specific cytotoxic T lymphocytes. We herein demonstrate that Mamu-B*098 samples lysophosphatidylethanolamine and lysophosphatidylcholine containing up to a C20 fatty acid in the ER. The X-ray crystal structures of Mamu-B*098 and Mamu-B*05104 complexed with lysophospholipids at high resolution revealed that the B and D pockets in the antigen-binding grooves of these MHC class I molecules accommodate these lipids through a monoacylglycerol moiety. Consistent with the capacity to bind cellular lipid ligands, these two MHC class I molecules did not require TAP function for cell-surface expression. Collectively, these results indicate that peptide- and lipopeptide-presenting MHC class I subsets use distinct sources of endogenous ligands., (© 2020 Shima et al.)

Allosteric regulation is protein activation by effector binding at a site other than the active site. Here, we show via X-ray structural analysis of gibberellin 2-oxidase 3 (GA2ox3), and auxin dioxygenase (DAO), that such a mechanism maintains hormonal homeostasis in plants. Both enzymes form multimers by interacting via GA 4 and indole-3-acetic acid (IAA) at their binding interface. Via further functional analyses we reveal that multimerization of these enzymes gradually proceeds with increasing GA 4 and IAA concentrations; multimerized enzymes have higher specific activities than monomer forms, a system that should favour the maintenance of homeostasis for these phytohormones. Molecular dynamic analysis suggests a possible mechanism underlying increased GA2ox3 activity by multimerization-GA 4 in the interface of oligomerized GA2ox3s may be able to enter the active site with a low energy barrier. In summary, homeostatic systems for maintaining GA and IAA levels, based on a common allosteric mechanism, appear to have developed independently.

Certain bacterial species target the polysaccharide glycosaminoglycans (GAGs) of animal extracellular matrices for colonization and/or infection. GAGs such as hyaluronan and chondroitin sulfate consist of repeating disaccharide units of uronate and amino sugar residues, and are depolymerized to unsaturated disaccharides by bacterial extracellular or cell-surface polysaccharide lyase. The disaccharides are degraded and metabolized by cytoplasmic enzymes such as unsaturated glucuronyl hydrolase, isomerase, and reductase. The genes encoding these enzymes are assembled to form a GAG genetic cluster. Here, we demonstrate the Streptococcus agalactiae phosphotransferase system (PTS) for import of unsaturated hyaluronan disaccharide. S. agalactiae NEM316 was found to depolymerize and assimilate hyaluronan, whereas its mutant with a disruption in the PTS genes included in the GAG cluster was unable to grow on hyaluronan, while retaining the ability to depolymerize hyaluronan. Using toluene-treated wild-type cells, the PTS activity for import of unsaturated hyaluronan disaccharide was significantly higher than that observed in the absence of the substrate. In contrast, the PTS mutant was unable to import unsaturated hyaluronan disaccharide, indicating that the corresponding PTS is the only importer of fragmented hyaluronan, which is suitable for PTS to phosphorylate the substrate at the C-6 position. This is distinct from Streptobacillus moniliformis ATP-binding cassette transporter for import of sulfated and non-sulfated fragmented GAGs without substrate modification. The three-dimensional structure of streptococcal EIIA, one of the PTS components, was found to contain a Rossman-fold motif by X-ray crystallization. Docking of EIIA with another component EIIB by modeling provided structural insights into the phosphate transfer mechanism. This study is the first to identify the substrate (unsaturated hyaluronan disaccharide) recognized and imported by the streptococcal PTS. The PTS and ABC transporter for import of GAGs shed light on bacterial clever colonization/infection system targeting various animal polysaccharides., Competing Interests: The authors have declared that no competing interests exist.

GH11 xylanase XynJ from Bacillus sp. strain 41M-1 has a β-jellyroll fold composed of eight β strands with a deep active-site cleft. We hypothesized that the thermostability of XynJ will increase if the flexibility of the β strands in the jellyroll structure is decreased without impairing activity. To verify this hypothesis, we introduced random mutations into Tyr13-Arg104 and Gly169-Tyr194, both of which are located in the β-jellyroll fold of XynJ, to construct a site saturation mutagenesis library. By screening 576 clones followed by site saturation mutation analysis of Thr82, T82A was selected as the most thermostable variant. In the hydrolysis of beechwood xylan at pH 7.8, the temperatures required to reduce initial activity by 50% in 15 min were 61 °C for the wild-type XynJ (WT) and 65 °C for T82A. The optimum hydrolysis temperatures were 60 °C for WT and 65 °C for T82A. There was little difference in the k cat and K m values and the pH dependence of activity between WT and T82A. Crystallographic analysis of WT and T82A revealed that thermostabilization by the T82A mutation might result from the removal of unfavorable van der Waals interactions. Thus, a highly thermostable XynJ variant was generated without impairing activity using this mutation strategy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Glycosaminoglycans (GAGs) such as hyaluronan and chondroitin in animal extracellular matrices contain disaccharide-repeating units. In a Gram-negative pathogenic Streptobacillus moniliformis , which belongs to Fusobacteria phylum and resides in rodent oral cavities, the solute-binding protein (Smon0123)-dependent ATP-binding cassette transporter imports unsaturated hyaluronan/chondroitin disaccharides into the cytoplasm after GAG lyase-dependent depolymerization. Here we show substrate recognition of unsaturated hyaluronan disaccharide by Smon0123. Moreover, Smon0123 exhibited no affinity for unsaturated chondroitin disaccharides containing three sulfate groups, distinct from non-sulfated, mono-sulfated, and di-sulfated chondroitin disaccharides previously identified as substrates. Crystal structure of Smon0123 with unsaturated hyaluronan disaccharide demonstrates that several residues, including Trp284 and Glu410, are crucial for binding to unsaturated hyaluronan/chondroitin disaccharides, whereas arrangements of water molecules at binding sites are found to be substrate dependent through comparison with substrate-bound structures determined previously. These residues are well conserved in Smon0123-like proteins of fusobacteria, and probably facilitate the fusobacterial residence in hyaluronan-rich oral cavities.

Klebsiella pneumoniae pullulanase (KPP) belongs to glycoside hydrolase family 13 subfamily 13 (GH13_13) and is the only enzyme that is reported to perform an induced-fit motion of the active-site loop (residues 706-710). Comparison of pullulanase structures indicated that only KPP has Leu680 present behind the loop, in contrast to the glycine found in other GH13_13 members. Analysis of the structure and activity of recombinant pullulanase from K. pneumoniae ATCC 9621 (rKPP) and its mutant (rKPP-G680L) indicated that the side chain of residue 680 is important for the induced-fit motion of the loop 706-710 and alters the binding affinity of the substrate.

Ceriporiopsis subvermispora (C. subvermispora) is a selective degrader of lignin in the woody biomass. Glutathione S-transferases (GSTs) are multifunctional enzymes that play important roles in cellular detoxification and metabolism. The crystal structures of a GST of C. subvermispora, CsGST83044, in GSH-free and -bound forms were solved at 1.95 and 2.19 Å resolution, respectively. The structure of the GSH-bound form revealed that CsGST83044 can be categorized as an atypical-type of GST. In the GSH-bound form of CsGST83044, Asn22, Asn24, and Tyr46 are located closest to the sulfur atom and form hydrogen bonds with the thiol group. The functional mutagenesis indicated that they are critical for the enzymatic activities of CsGST83044. The critical residues of an atypical-type GST belonging to the GSTFuA class were revealed for the first time. A previous study indicated that CsGST83044 and another GST, CsGST63524, differ in substrate preference; CsGST83044 prefers smaller substrates than CsGST63524 for its esterase activity. The GSH-bound pocket of CsGST83044 turns out to be small, which may explain the preference for smaller substrates. Protein engineering of GSTs of C. subvermispora in the light of the obtained insight may pave a path in the future for utilization of the woody biomass., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aspergillus oryzae RIB40 formate oxidase has Arg87 and Arg554 near the formyl group and O(4) atom of 8-formyl-flavin adenine dinucleotide (FAD), respectively, with Asp396 neighbouring Arg554. Herein, we probed the roles of these three residues in modification of FAD to 8-formyl-FAD. Replacement of Arg87 or Arg554 with Lys or Ala decreased and abolished the modification, respectively. Replacement of Asp396 with Ala or Asn lowered the modification rate. The observation of unusual effects of maintaining pH 7.0 on the modification in R87K, R554K and D396 variants indicates initial and subsequent processes with different pH dependencies. Comparison of the initial process at pH 4.5 and 7.0 suggests that the microenvironment around Arg87 and the protonation state of Asp396 affect the initial process in the native enzyme. Comparison of the crystal structures of native and R554 variants showed that the replacements had minimal effect on catalytic site structure. The positively charged Arg87 might contribute to the formation of an anionic quinone-methide tautomer intermediate, while the positively charged Arg554, in collaboration with the negatively charged Asp396, might stabilize this intermediate and form a hydrogen bonding network with the N(5)/O(4) region, thereby facilitating efficient FAD modification., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Similar to host proteins, N -myristoylation occurs for viral proteins to dictate their pathological function. However, this lipid-modifying reaction creates a novel class of "lipopeptide" Ags targeted by host CTLs. The primate MHC class I-encoded protein, Mamu-B*098, was previously shown to bind N -myristoylated 5-mer peptides. Nevertheless, T cells exist that recognize even shorter lipopeptides, and much remains to be elucidated concerning the molecular mechanisms of lipopeptide presentation. We, in this study, demonstrate that the MHC class I allele, Mamu-B*05104, binds the N -myristoylated 4-mer peptide (C14-Gly-Gly-Ala-Ile) derived from the viral Nef protein for its presentation to CTLs. A phylogenetic tree analysis indicates that these classical MHC class I alleles are not closely associated; however, the high-resolution x-ray crystallographic analyses indicate that both molecules share lipid-binding structures defined by the exceptionally large, hydrophobic B pocket to accommodate the acylated glycine (G1) as an anchor. The C-terminal isoleucine (I4) of C14-Gly-Gly-Ala-Ile anchors at the F pocket, which is distinct from that of Mamu-B*098 and is virtually identical to that of the peptide-presenting MHC class I molecule, HLA-B51. The two central amino acid residues (G2 and A3) are only exposed externally for recognition by T cells, and the methyl side chain on A3 constitutes a major T cell epitope, underscoring that the epitopic diversity is highly limited for lipopeptides as compared with that for MHC class I-presented long peptides. These structural features suggest that lipopeptide-presenting MHC class I alleles comprise a distinct MHC class I subset that mediates an alternative pathway for CTL activation., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Although endogenous animal cellulases have great potential for industrial applications such as bioethanol production, few investigations have focused on these enzymes. In this study, the glycoside hydrolase family 45 (GH45) subfamily B endoglucanase EG27II from the snail Ampullaria crossean was expressed using a Pichia pastoris expression system and the crystal structure of the apo form was determined at 1.00 Å resolution; this is the highest resolution structure of an animal endoglucanase. The results showed that EG27II has a double-ψ six-stranded β-barrel and that the structure of EG27II more closely resembles those of subfamily C enzymes than those of subfamily A enzymes. The structure of EG27II complexed with cellobiose was also determined under cryoconditions and at room temperature at three pH values, pH 4.0, 5.5 and 8.0, and no structural changes were found to be associated with the change in pH. The structural comparison and catalytic activity measurements showed that Asp137 and Asn112 function as the catalytic acid and base, respectively, and that Asp27 is also an important residue for catalysis. These high-resolution structures of EG27II provide a large amount of information for structure-based enzyme modification and cell-surface engineering, which will advance biofuel production using animal-derived cellulases.

Ceriporiopsis subvermispora (C. subvermispora), one of the white-rot fungi, is known as a selective lignin degrader of the woody biomass. Glutathione S-transferases (GSTs) are multifunctional enzymes that are capable of catalyzing the reactions involved in detoxification and metabolic pathways. In this study, a GST of C. subvermispora, named CsGST63524, was overexpressed in E. coli, and then purified by affinity, anion exchange, and size exclusion column chromatography. The crystal structures of the CsGST63524 in ligand-free and complex with GSH were refined at 2.45 and 2.50 Å resolutions, respectively. The sulfur atom of glutathione forms a hydrogen bond with Ser21 of CsGST63524, indicating it is a serine-type GST. Mutagenesis of Ser21 unexpectedly indicated that this serine residue is not essential for the enzymatic activity of CsGST63524. Comparative sequence and structural analyses, together with functional mutagenesis, newly identified the enzymatically important non-canonical amino acid residues, Asn23 and Tyr45, other than the serine residue., (Copyright © 2019 Elsevier Inc. All rights reserved.)

One of the sweetest proteins found in tropical fruits (with a threshold of 50 nM), thaumatin, is also used commercially as a sweetener. Our previous study successfully produced the sweetest thaumatin mutant (D21N), designated hyper-sweet thaumatin, which decreases the sweetness threshold to 31 nM. To investigate why the D21N mutant is sweeter than wild-type thaumatin, we compared the structure of the D21N mutant solved at a subatomic resolution of 0.93 Å with that of wild-type thaumatin determined at 0.90 Å. Although the overall structure of the D21N mutant resembles that of wild-type thaumatin, our subatomic resolution analysis successfully assigned and discriminated the detailed atomic positions of side-chains at position 21. The relative B-factor value of the side-chain at position 21 in the D21N mutant was higher than that of wild-type thaumatin, hinting at a greater flexibility of side-chain at 21 in the hyper-sweet D21N mutant. Furthermore, alternative conformations of Lys19, which is hydrogen-bonded to Asp21 in wild-type, were found only in the D21N mutant. Subatomic resolution analysis revealed that flexible conformations at the sites adjacent to positions 19 and 21 play a crucial role in enhancing sweet potency and may serve to enhance the complementarity of electrostatic potentials for interaction with the sweet taste receptor., (Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Crystal structures of Klebsiella pneumoniae pullulanase (KPP) in complex with α-cyclodextrin (α-CD), β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD) were refined at around 1.98-2.59 Å resolution from data collected at SPring-8. In the structures of the complexes obtained with 1 mM α-CD or γ-CD, one molecule of CD was found at carbohydrate-binding module 41 only (CBM41). In the structures of the complexes obtained with 1 mM β-CD or with 10 mM α-CD or γ-CD, two molecules of CD were found at CBM41 and in the active-site cleft, where the hydrophobic residue of Phe746 occupies the inside cavity of the CD rings. In contrast to α-CD and γ-CD, one β-CD molecule was found at the active site only in the presence of 0.1 mM β-CD. These results were coincident with the solution experiments, which showed that β-CD inhibits this enzyme more than a thousand times more potently than α-CD and γ-CD. The strong inhibition of β-CD is caused by the optimized interaction between β-CD and the side chain of Phe746. The increased K i values of the F746A mutant for β-CD supported the importance of Phe746 in the strong interaction of pullulanase with β-CD.

Ferritin, a ubiquitous iron storage protein, has a crucial role in innate immunity in arthropods, which have no adaptive immune system. Arthropods are thought to have two types of ferritin molecules: the secreted type and the cytosolic type. Here, we present the first crystal structure of ferritin from crustacean, kuruma prawn (Marsupenaeus japonicus), at 1.16 Å resolution. This shrimp ferritin (MjFer) is the cytosolic type, and its structure shows well-conserved ferritin fold composed of a 4-helix bundle that assembles into a cage-like 24-mer. The structure of MjFer was more similar to those of human and vertebrate ferritins than to that of the secreted-type arthropod ferritin from an insect. MjFer possesses both a ferroxidase site and a nucleation site, which are the main characteristics of vertebrate H and L chain ferritins, respectively. The first crystal structure of crustacean ferritin, MjFer, has exceptionally high quality that provides the detailed structural information of metal moving pathway in ferritin., (© 2018 The Protein Society.)

Hetero-trimeric aldehyde oxidases of bacterial origin, which use O2 to catalyse the oxidation of various aldehydes but not those of aromatic N-heterocycles, belong to the xanthine oxidase family. In the present study, the crystal structure of a recombinant aldehyde oxidase from Methylobacillus sp. KY4400 (Mb-AOX) was determined at 2.5 Å resolution. The structures of its subunits resemble those of the corresponding subunits or domains of other structurally characterised enzymes belonging to the family, and include a [4Fe-4 S] cluster in the medium subunit like that found in Escherichia coli periplasmic aldehyde oxidoreductase (EP-AOR). A funnel leading to the si-face of the isoalloxazine ring of FAD, which is narrower than those in mouse liver AOX3 and human AOX1, is also present and it is even narrower than that in EP-AOR. The environment surrounding the ring in Mb-AOX and EP-AOR is subtly different, which might account for their different abilities to use O2. A remarkable characteristic of the Mo catalytic centre in Mb-AOX is a tryptophan situated near the centre instead of the alanine present in other xanthine oxidase family members. The tryptophan residue together with other residue differences might play an important role in binding to aldehydes such as n-heptylaldehyde in Mb-AOX.

Endo-1,3-β-glucanase from Cellulosimicrobium cellulans is composed of a catalytic domain and a carbohydrate-binding module. We have determined the X-ray crystal structure of the catalytic domain at a high resolution of 1.66Å. The overall fold is a sandwich-like β-jelly roll architecture like the enzymes in the glycoside hydrolase family 16. The substrate-binding cleft has a length and a width of ~28 and ~15Å, respectively, which is thought to be capable of accommodating at least six glucopyranose units. Laminarihexaose was placed into the substrate-binding cleft, namely at the subsites +2 to -4 from the reducing end, and the complex structure was analyzed using molecular dynamics simulations (MD) and using a rotamer search of the pocket. During the MD simulations, the substrate fluctuated more than the enzyme, where the residues at the subsites toward the non-reducing end fluctuated more than those toward the reducing end. Little conformational change of the protein was observed for the subsites +1 and +2, indicating that the glucose's position could be tightly restricted inside the pocket. Substrate binding experiments using isothermal titration calorimetry showed that the binding affinity of laminaritriose was higher than that of laminaribiose and similar to those of other longer laminarioligosaccharides. Taken together, the substrates mainly bind to the subsites -1 to -3 with the highest affinity, while the part bound to the reducing end would be hydrolyzed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Thaumatin, an intensely sweet-tasting protein, elicits sweet taste with a threshold of only 50 nM. Previous studies from our laboratory suggested that the complex model between the T1R2-T1R3 sweet receptor and thaumatin depends critically on the complementarity of electrostatic potentials. In order to further validate this model, we focused on three lysine residues (Lys78, Lys106, and Lys137), which were expected to be part of the interaction sites. Three thaumatin mutants (K78A, K106A, and K137A) were prepared and their threshold values of sweetness were examined. The results showed that the sweetness of K106A was reduced by about three times and those of K78A and K137A were reduced by about five times when compared to wild-type thaumatin. The three-dimensional structures of these mutants were also determined by X-ray crystallographic analyses at atomic resolutions. The overall structures of mutant proteins were similar to that of wild-type but the electrostatic potentials around the mutated sites became more negative. Since the three lysine residues are located in 20-40 Å apart each other on the surface of thaumatin molecule, these results suggest the positive charges on the surface of thaumatin play a crucial role in the interaction with the sweet receptor, and are consistent with a large surface is required for interaction with the sweet receptor, as proposed by the multipoint interaction model named wedge model.

Glycosaminoglycans (GAGs), constituted by repeating uronate and amino sugar units, are major components of mammalian extracellular matrices. Some indigenous and pathogenic bacteria target GAGs for colonization to and/or infection of host mammalian cells. In Gram-negative pathogenic Streptobacillus moniliformis, the solute-binding protein (Smon0123)-dependent ATP-binding cassette (ABC) transporter incorporates unsaturated GAG disaccharides into the cytoplasm after depolymerization by polysaccharide lyase. Smon0123, composed of N and C domains, adopts either a substrate-free open or a substrate-bound closed form by approaching two domains at 47° in comparison with the open form. Here we show an alternative 39°-closed conformation of Smon0123 bound to unsaturated chondroitin disaccharide sulfated at the C-4 and C-6 positions of N-acetyl-d-galactosamine residue (CΔ4S6S). In CΔ4S6S-bound Smon0123, Arg204 and Lys210 around the two sulfate groups were located at different positions from those at other substrate-bound 47°-closed conformations. Therefore, the two sulfate groups in CΔ4S6S shifted substrate-binding residue arrangements, causing dynamic conformational change. Smon0123 showed less affinity with CΔ4S6S than with non-sulfated and monosulfated substrates. ATPase activity of the Smon0123-dependent ABC transporter in the presence of CΔ4S6S was lower than that in the presence of other unsaturated chondroitin disaccharides, suggesting that CΔ4S6S-bound Smon0123 was unpreferable for docking with the ABC transporter.

The tripartite EfeUOB system functions as a low pH iron importer in Gram-negative bacteria. In the alginate-assimilating bacterium Sphingomonas sp. strain A1, an additional EfeO-like protein (Algp7) is encoded downstream of the efeUOB operon. Here we show the metal binding mode of Algp7, which carries a M_75 metallopeptidase motif. The Algp7 protein was purified from recombinant E. coli cells and was subsequently characterized using differential scanning fluorimetry, fluorescence spectrometry, atomic absorption spectroscopy, and X-ray crystallography. The fluorescence of a dye, SYPRO Orange, bound to denatured Algp7 in the absence and presence of metal ions was measured during heat treatment. The fluorescence profile of Algp7 in the presence of metals such as ferric, ferrous, and zinc ions, shifted to a higher temperature, suggesting that Algp7 binds these metal ions and that metal ion-bound Algp7 is more thermally stable than the ligand-free form. Algp7 was directly demonstrated to show an ability to bind copper ion by atomic absorption spectroscopy. Crystal structure of metal ion-bound Algp7 revealed that the metal ion is bound to the cleft surrounded by several acidic residues. Four residues, Glu79, Glu82, Asp96, and Glu178, distinct from the M_75 motif (His115xxGlu118), are coordinated to the metal ion. This is the first report to provide structural insights into metal binding by the bacterial EfeO element., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The Gram-negative bacterium Sphingomonas sp. A1 incorporates alginate into cells via the cell-surface pit without prior depolymerization by extracellular enzymes. Alginate import across cytoplasmic membranes thereby depends on the ATP-binding cassette transporter AlgM1M2SS (a heterotetramer of AlgM1, AlgM2, and AlgS), which cooperates with the periplasmic solute-binding protein AlgQ1 or AlgQ2; however, several details of AlgM1M2SS-mediated alginate import are not well-understood. Herein, we analyzed ATPase and transport activities of AlgM1M2SS after reconstitution into liposomes with AlgQ2 and alginate oligosaccharide substrates having different polymerization degrees (PDs). Longer alginate oligosaccharides (PD ≥ 5) stimulated the ATPase activity of AlgM1M2SS but were inert as substrates of AlgM1M2SS-mediated transport, indicating that AlgM1M2SS-mediated ATP hydrolysis can be stimulated independently of substrate transport. Using X-ray crystallography in the presence of AlgQ2 and long alginate oligosaccharides (PD 6-8) and with the humid air and glue-coating method, we determined the crystal structure of AlgM1M2SS in complex with oligosaccharide-bound AlgQ2 at 3.6 Å resolution. The structure of the ATP-binding cassette transporter in complex with non-transport ligand-bound periplasmic solute-binding protein revealed that AlgM1M2SS and AlgQ2 adopt inward-facing and closed conformations, respectively. These in vitro assays and structural analyses indicated that interactions between AlgM1M2SS in the inward-facing conformation and periplasmic ligand-bound AlgQ2 in the closed conformation induce ATP hydrolysis by the ATP-binding protein AlgS. We conclude that substrate-bound AlgQ2 in the closed conformation initially interacts with AlgM1M2SS, the AlgM1M2SS-AlgQ2 complex then forms, and this formation is followed by ATP hydrolysis., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Glycosaminoglycans (GAGs), such as hyaluronan, chondroitin sulfate, and heparin, constitute mammalian extracellular matrices. The uronate and amino sugar residues in hyaluronan and chondroitin sulfate are linked by 1,3-glycoside bond, while heparin contains 1,4-glycoside bond. Some bacteria target GAGs as means of establishing colonization and/or infection, and bacterial degradation mechanisms of GAGs have been well characterized. However, little is known about the bacterial import of GAGs. Here, we show a GAG import system, comprised of a solute-binding protein (Smon0123)-dependent ATP-binding cassette (ABC) transporter, in the pathogenic Streptobacillus moniliformis. A genetic cluster responsible for depolymerization, degradation, and metabolism of GAGs as well as the ABC transporter system was found in the S. moniliformis genome. This bacterium degraded hyaluronan and chondroitin sulfate with an expression of the genetic cluster, while heparin repressed the bacterial growth. The purified recombinant Smon0123 exhibited an affinity with disaccharides generated from hyaluronan and chondroitin sulfate. X-ray crystallography indicated binding mode of Smon0123 to GAG disaccharides. The purified recombinant ABC transporter as a tetramer (Smon0121-Smon0122/Smon0120-Smon0120) reconstructed in liposomes enhanced its ATPase activity in the presence of Smon0123 and GAG disaccharides. This is the first report that has molecularly depicted a bacterial import system of both sulfated and non-sulfated GAGs.

Atomic resolution structures (beyond 1.20 Å) at ambient temperature, which is usually hampered by the radiation damage in synchrotron X-ray crystallography (SRX), will add to our understanding of the structure-function relationships of enzymes. Serial femtosecond crystallography (SFX) has attracted surging interest by providing a route to bypass such challenges. Yet the progress on atomic resolution analysis with SFX has been rather slow. In this report, we describe the 1.20 Å resolution structure of proteinase K using 13 keV photon energy. Hydrogen atoms, water molecules, and a number of alternative side-chain conformations have been resolved. The increase in the value of B-factor in SFX suggests that the residues and water molecules adjacent to active sites were flexible and exhibited dynamic motions at specific substrate-recognition sites.

The flavoenzyme 2-Methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO) catalyzes the cleavage of the pyridine ring of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) in the presence of NADH, molecular oxygen, and water. MHPCO also catalyzes the NADH oxidation reaction uncoupled with ring opening in the absence of MHPC (the basal activity). The enzyme shows activity toward not only MHPC but also 5-hydroxynicotinic acid (5HN) and 5-pyridoxic acid (5PA). The reaction rate toward 5PA is extremely low (5% of the activity toward MHPC or 5HN). We determined the crystal structures of MHPCO without substrate and the MHPCO/5HN and MHPCO/5PA complexes, together with a Y270F mutant without substrate and its 5HN complex. The Tyr270 residue was located in the active site and formed hydrogen bonds between the Oη and water molecules to make the active site hydrophilic. Although Tyr270 took a fixed conformation in the structures of the MHPCO and MHPCO/5HN complex, it took two conformations in its 5PA complex, accompanied by two conformations of the bound 5PA. In the wild-type (WT) enzyme, the turnover number of the ring-opening activity was 6800 times that of the basal activity (1300 and 0.19 s -1 , respectively), whereas no such difference was observed in the Y270F (19 and 7.4 s -1 ) or Y270A (0.05 and 0.84 s -1 ) mutants. In the Y270F/5HN complex, the substrate bound ∼1 Å farther away than in the WT enzyme. These results revealed that Tyr270 is essential to maintain the WT conformation, which in turn enhances the coupling of the NADH oxidation with the ring-opening reaction., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)