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The rising prevalence of antimicrobial resistance (AMR) of bacteria is a global health problem at the human, animal, and environmental interfaces, which necessitates the "One Health" approach. AMR of bacteria in animal feed are a potential cause of the prevalence in livestock; however, the role remains unclear. To date, there is limited research on AMR of bacteria in animal feed in Japan. In this study, a total of 57 complete feed samples and 275 feed ingredient samples were collected between 2018 and 2020. Enterococcus spp. were present in 82.5% of complete feed (47/57 samples), 76.5% of soybean meal (62/81), 49.6% of fish meal (55/111), 33.3% of poultry meal (22/66), and 47.1% of meat and bone meal (8/17) samples. Of 295 isolates, E. faecium (33.2% of total isolates) was the dominant Enterococcus spp., followed by E. faecalis (14.2%), E. hirae (6.4%), E. durans (2.7%), E. casseliflavus (2.4%), and E. gallinarum (1.0%). Of 134 isolates which were tested for antimicrobial susceptibility, resistance to kanamycin was the highest (26.1%), followed by erythromycin (24.6%), tetracycline (6.0%), lincomycin (2.2%), tylosin (1.5%), gentamicin (0.8%), and ciprofloxacin (0.8%). All Enterococcus spp. exhibited susceptibility to ampicillin, vancomycin, and chloramphenicol. Of 33 erythromycin-resistant isolates, only two showed a high minimum inhibitory concentration value (>128 μg/mL) and possessed ermB . These results revealed that overall resistance to antimicrobials is relatively low; however, animal feed is a source of Enterococcus spp. It is essential to elucidate the causative factors related to the prevalence of AMR in animal feed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yamagami, Asao, Takahashi, Hashimoto, Okuyama, Arai, Arihara, Masui and Shimazaki.)

Transketolase is a key enzyme in the pentose phosphate pathway in all organisms, recognizing sugar phosphates as substrates. Transketolase with a cofactor of thiamine pyrophosphate catalyzes the transfer of a 2-carbon unit from D-xylulose-5-phosphate to D-ribose-5-phosphate (5-carbon aldose), giving D-sedoheptulose-7-phosphate (7-carbon ketose). Transketolases can also recognize non-phosphorylated monosaccharides as substrates, and catalyze the formation of non-phosphorylated 7-carbon ketose (heptulose), which has attracted pharmaceutical attention as an inhibitor of sugar metabolism. Here, we report the structural and biochemical characterizations of transketolase from Thermus thermophilus HB8 (TtTK), a well-characterized thermophilic Gram-negative bacterium. TtTK showed marked thermostability with maximum enzyme activity at 85 °C, and efficiently catalyzed the formation of heptuloses from lithium hydroxypyruvate and four aldopentoses: D-ribose, L-lyxose, L-arabinose, and D-xylose. The X-ray structure showed that TtTK tightly forms a homodimer with more interactions between subunits compared with transketolase from other organisms, contributing to its thermal stability. A modeling study based on X-ray structures suggested that D-ribose and L-lyxose could bind to the catalytic site of TtTK to form favorable hydrogen bonds with the enzyme, explaining the high conversion rates of 41% (D-ribose) and 43% (L-lyxose) to heptulose. These results demonstrate the potential of TtTK as an enzyme producing a rare sugar of heptulose. KEY POINTS: • Transketolase catalyzes the formation of a 7-carbon sugar phosphate • Structural and biochemical characterizations of thermophilic transketolase were done • The enzyme could produce non-phosphorylated 7-carbon ketoses from sugars., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Fatty acid kinase is necessary for the incorporation of exogenous fatty acids into membrane phospholipids. Fatty acid kinase consists of two components: a kinase component, FakA, that phosphorylates a fatty acid bound to a fatty acid-binding component, FakB. However, the molecular details underlying the phosphotransfer reaction remain to be resolved. We determined the crystal structure of the N-terminal domain of FakA bound to ADP from Thermus thermophilus HB8. The overall structure of this domain showed that the helical barrel fold is similar to the nucleotide-binding component of dihydroxyacetone kinase. The structure of the nucleotide-binding site revealed the roles of the conserved residues in recognition of ADP and Mg 2+ , but the N-terminal domain of FakA lacked the ADP-capping loop found in the dihydroxyacetone kinase component. Based on the structural similarity to the two subunits of dihydroxyacetone kinase complex, we constructed a model of the complex of T. thermophilus FakB and the N-terminal domain of FakA. In this model, the invariant Arg residue of FakB occupied a position that was spatially similar to that of the catalytically important Arg residue of dihydroxyacetone kinase, which predicted a composite active site in the Fatty acid kinase complex., 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.)

Thermophilic proteins maintain their structure at high temperatures through a combination of various factors. Here, we report the ligand-induced stabilization of a thermophilic Ser/Thr protein kinase. Thermus thermophilus TpkD unfolds completely at 55 °C despite the optimum growth temperature of 75 °C. Unexpectedly, we found that the TpkD structure is drastically stabilized by its natural ligands ATP and ADP, as evidenced by the increase in the melting temperature to 80 °C. Such a striking effect of a substrate on thermostability has not been reported for other protein kinases. Conformational changes upon ATP binding were observed in fluorescence quenching and limited proteolysis experiments. Urea denaturation of Trp mutants suggested that ATP binding affects not only the ATP-binding site, but also the remote regions. Our findings shed light on thermoadaptation of thermophilic proteins., (© 2020 Federation of European Biochemical Societies.)

Animal carcinogenesis models induced by environmental chemicals have been widely used for basic and applied cancer research. However, establishment of in vitro or ex vivo models is essential for molecular mechanistic elucidation of early events in carcinogenesis, leading to clarification of the total mode of action. In the present study, to establish an organoid-based chemical carcinogenesis model, mouse organoids were treated in vitro with 4 genotoxic chemicals, e.g. ethyl methanesulfonate (EMS), acrylamide (AA), diethylnitrosamine (DEN) and 7,12-dimethylbenz[a]anthracene (DMBA) to examine their tumorigenicity after injection to nude mice. The four chemicals were reported to induce lung, liver or mammary carcinomas in mouse models. DMBA-treated mammary tissue-derived organoids with Trp53 heterozygous knockout exhibited tumorigenicity, but not those with wild-type Trp53, reflecting previous reports of corresponding animal models. Treatment of lung organoids with or without Trp53 knockout with EMS or AA resulted in carcinogenic histopathological characteristics, and the activation of oncogenic kinases was demonstrated in the nodules from the nude mouse subcutis. DEN-treated liver (biliary tract) organoids also had an increased number of similar changes. In conclusion, an ex vivo model for chemical carcinogenesis was established using normal mouse tissue-derived organoids. This model will be applied to detect early molecular events, leading to clarification of the mode of action of chemical carcinogenesis., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Agarwood has been used as an incense and in traditional medicines as aphrodisiac, sedative, cardiotonic, and carminative. In this study, five new 2-(2-phenylethyl)chromones (2, 13-16) and eleven known compounds (1, 3-12) were isolated from the agarwood. The structures of the new compounds were determined by 1 H-, 13 C-, and two-dimensional NMR together with electronic circular dichroism (ECD) spectroscopy. All isolated compounds were evaluated for the phosphodiesterase (PDE) 3A and 5A1 inhibitory activity by the fluorescence polarization method. Dimeric 2-(2-phenylehyl)chromones (13, 14, 16) had potent inhibitory activity to PDE 5A1 with IC 50 values of micro molar range (13: 4.2 μM, 14: 7.9 μM, 16: 4.3 μM), whereas they had weak activity to PDE 3A. In contrast, compound (15), which has a phenylpropionic acid moiety instead of the 2-(2-phenylethyl)chromone moiety in the dimers, showed moderate inhibition of both PDE 3A (IC 50 : 42.6 μM) and PDE 5A1 (IC 50 : 15.1 μM).

Nucleoside monophosphate kinases play crucial roles in biosynthesis and regeneration of nucleotides. These are bi-substrate enzymes that catalyze reversible transfers of a phosphoryl group between ATP and nucleoside monophosphate. These enzymes are comprised of the CORE domain, the NMP-binding domain, and the LID domain. Large conformational rearrangement of the three domains occurs during the catalytic cycle. Although many structures of CMP kinase have been determined, only limited structural information has been available on the conformational changes along the reaction pathway. We determined five crystal structures of CMP kinase of Thermus thermophilus HB8 in ligand-free form and the CMP "open", CMP "closed", ADP-CDP-Gd3+-, and CDP-bound forms at resolutions of 1.7, 2.2, 1.5, 1.6, and 1.7 Å, respectively. The ligand-free form was in an open conformation, whereas the structures of the CMP "closed", ADP-CDP-Gd3+-, and CDP-bound forms were in a closed conformation, in which the shift of the NMP-binding domain and LID domain caused closure of the substrate-binding cleft. Interestingly, the CMP "open" form was in an open conformation even with CMP bound, implying intrinsic conformational fluctuation. The structure of the ADP-CDP complex is the first structure of CMP kinase with a phosphoryl group donor and an acceptor. Upon simultaneous binding of ADP and CDP, the side chains of several residues in the LID domain moved toward the nucleotides without global open-closed conformational changes compared to those in the CMP "closed" and CDP complexes. These global and local conformational changes may be crucial for the substrate recognition and catalysis. The terminal phosphate groups of ADP and CDP had similar geometry to those of two ADP in AMP kinase, suggesting common catalytic mechanisms to other nucleoside monophosphate kinases. Our findings are expected to contribute to detailed understanding of the reaction mechanism of CMP kinase., Competing Interests: The authors have declared that no competing interests exist.

The primary pharmacy system and health support pharmacy system were established in 2016. However, local pharmacies need to get closer to the community. To this end, each pharmacy is making efforts to contribute locally. Here, we introduce various initiatives in our region. Akakabe Pharmacy has 66 stores in Osaka Prefecture, mainly in the northeastern part of Osaka, where the elderly population is growing. We are implementing a dominant strategy: cooperation with the city and administration is strong, and we hold many related events directed towards the public. For example, two thousand participants gathered in an event sponsored by the city aimed at the improvement of beauty and health. At such events, participants can easily consult with pharmacists. Dispensing pharmacy stores-pharmacies that combine the features of a convenience store with care consulting services-were established in 2016. Care consultations are potentially highly advantageous to the users. In the consultation space of a pharmacy, a care worker conducts various events every month, such as on dementia prevention, body composition measurement, and more. We believe that this type of combined pharmacy and convenience store has the potential to become a regional comprehensive care center. We intend to share the possibility of a new pharmacy system, centered on this pharmacy/store/consultation model, as a basis to revamp the pharmacy industry.

NurA and HerA are thought to be essential proteins for DNA end resection in archaeal homologous recombination systems. Thermus thermophilus , an extremely thermophilic eubacterium, has proteins that exhibit significant sequence similarity to archaeal NurA and HerA. To unveil the cellular function of NurA and HerA in T. thermophilus , we performed phenotypic analysis of disruptant mutants of nurA and herA with or without DNA-damaging agents. The nurA and herA genes were not essential for survival, and their deletion had no effect on cell growth and genome integrity. Unexpectedly, these disruptants of T. thermophilus showed increased resistance to UV irradiation and mitomycin C treatment. Further, these disruptants and the wild type displayed no difference in sensitivity to oxidative stress and a DNA replication inhibitor. T. thermophilus NurA had nuclease activity, and HerA had ATPase. The overexpression of loss-of-function mutants of nurA and herA in the respective disruptants showed no complementation, suggesting their enzymatic activities were involved in the UV sensitivity. In addition, T. thermophilus NurA and HerA interacted with each other in vitro and in vivo , forming a complex with 2:6 stoichiometry. These results suggest that the NurA-HerA complex has an architecture similar to that of archaeal counterparts but that it impairs, rather than promotes, the repair of photoproducts and DNA cross-links in T. thermophilus cells. This cellular function is distinctly different from that of archaeal NurA and HerA. IMPORTANCE Many nucleases and helicases are engaged in homologous recombination-mediated DNA repair. Previous in vitro analyses in archaea indicated that NurA and HerA are the recombination-related nuclease and helicase. However, their cellular function had not been fully understood, especially in bacterial cells. In this study, we performed in vivo analyses to address the cellular function of nurA and herA in an extremely thermophilic bacterium, Thermus thermophilus As a result, T. thermophilus NurA and HerA exhibited an interfering effect on the repair of several instances of DNA damage in the cell, which is in contrast to the results in archaea. This finding will facilitate our understanding of the diverse cellular functions of the recombination-related nucleases and helicases., (Copyright © 2018 American Society for Microbiology.)

Agarwood (jinkoh in Japanese) is a resinous wood from Aquilaria species of the family Thymelaeaceae and has been used as incense and in traditional medicines. Characteristic chromone derivatives such as agarotetrol have been isolated from agarwood. In previous study, we isolated two new 2-(2-phenylethyl)chromones together with six known compounds from MeOH extract of agarwood. Further chemical investigation of the MeOH extract led to isolation of eighteen 2-(2-phenylethyl)chromones, including three new 5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromones with stereochemistry enantiomeric to agarotetrol-type, viz. (5R,6S,7S,8R)-2-[2-(3'-hydroxy-4'-methoxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone (2), (5R,6S,7S,8R)-2-[2-(4'-methoxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone (6), and (5R,6S,7S,8R)-2-[2-(4'-hydroxy-3'- methoxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone (13). The absolute configurations of the new compounds were determined by exciton chirality method. All isolated compounds were tested for their phosphodiesterase (PDE) 3A inhibitory activity by fluorescence polarization method. Compounds 8, 12-15, 21-24 showed moderate PDE 3A inhibitory activity.

Saffron, the stigma of Crocus sativus Linné (Iridaceae family), has been known to inhibit aggregation of β-amyloid, a nerve tissue protein. α-Synuclein (αS) is a 140-amino acid protein found abundantly in various regions of the brain. Its abnormal aggregation and accumulation in nerve tissue are said to cause neurodegenerative diseases such as Parkinson's disease, Lewy body dementia, and multiple-system atrophy. This study (part of this study was presented at the 137th Annual Meeting of the Pharmaceutical Society of Japan) examined the effects of saffron, its constituents (crocin-1, crocin-2, crocetin, and safranal), and crocetin structural analogs (hexadecanedioic acid, norbixin, and trans, trans-muconic acid) on αS aggregation, and αS fibril dissociation. Saffron dose-dependently inhibited αS aggregation and dissociated αS fibrils by thioflavin T fluorescence assay. These effects were observed by transmission electron microscopy, which showed reduced and shortened αS fibrils. Crocin-1, crocin-2, and crocetin showed anti-aggregation and fibril dissociation effects, with crocetin being the most potent. The effects of norbixin were weaker than those of crocetin, and the other crocetin structural analogs showed no effects. These results show that saffron and its constituents (crocin-1, crocin-2, and crocetin) can be effective in preventing and treating diseases caused by abnormal αS aggregation.

Uridine-cytidine kinase (UCK), including human UCK2, are a family of enzymes that generally phosphorylate both uridine and cytidine. However, UCK of Thermus thermophilus HB8 (ttCK) phosphorylates only cytidine. This cytidine-restricted activity is thought to depend on Tyr93, although the precise mechanism remains unresolved. Exhaustive mutagenesis of Tyr93 in ttCK revealed that the uridine phosphorylation activity was restored only by replacement of Tyr93 with His or Gln. Replacement of His117 in human UCK2, corresponding to residue Tyr93 in ttCK, by Tyr resulted in a loss of uridine phosphorylation activity. These findings indicated that uridine phosphorylation activity commonly depends on a single residue in the UCK family.

Homologous recombination (HR) plays an essential role in the maintenance of genome integrity. RecA/Rad51 paralogs have been recognized as an important factor of HR. Among them, only one bacterial RecA/Rad51 paralog, RadA, is involved in HR as an accessory factor of RecA recombinase. RadA has a unique Lon protease-like domain (LonC) at its C terminus, in addition to a RecA-like ATPase domain. Unlike Lon protease, RadA's LonC domain does not show protease activity but is still essential for RadA-mediated DNA repair. Reconciling these two facts has been difficult because RadA's tertiary structure and molecular function are unknown. Here, we describe the hexameric ring structure of RadA's LonC domain, as determined by X-ray crystallography. The structure revealed the two positively charged regions unique to the LonC domain of RadA are located at the intersubunit cleft and the central hole of a hexameric ring. Surprisingly, a functional domain analysis demonstrated the LonC domain of RadA binds DNA, with site-directed mutagenesis showing that the two positively charged regions are critical for this DNA-binding activity. Interestingly, only the intersubunit cleft was required for the DNA-dependent stimulation of ATPase activity of RadA, and at least the central hole was essential for DNA repair function. Our data provide the structural and functional features of the LonC domain and their function in RadA-mediated DNA repair., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Recent studies have revealed the physiological significance of post-translational lysine acylations such as acetylation in the regulation of various cellular processes. Here, we characterized lysine propionylation, a recently discovered post-translational acylation, in five representative bacteria: Geobacillus kaustophilus, Thermus thermophilus, Escherichia coli, Bacillus subtilis, and Rhodothermus marinus. Using antibody-based propionyl peptide enrichment followed by identification with nano-liquid chromatography tandem mass spectrometry, we showed that proteins were subject to lysine propionylation in all five bacterial species analyzed. Notably, many propionylations were identified in the Bacillus-related, thermophilic eubacterium G. kaustophilus, but fewer in the mesophilic eubacterium B. subtilis, suggesting that propionylation event abundance is independent of phylogenetic relationship. We further found propionylation sites in the thermophilic eubacterium T. thermophilus, but the thermophilic eubacterium R. marinus showed the fewest number of sites, indicating that growth temperature is not a determinant of propionylation state. In silico analyses demonstrated that lysine propionylation is related to metabolic pathways, particularly those controlled by acyl-CoA synthetases, similar to lysine acetylation. We also detected dozens of propionylation sites at positions important for protein functions across bacteria, demonstrating the regulatory mechanisms affected by lysine propionylations. Our proteome-wide analyses across bacteria thus provide insights into the general functions of lysine propionylation.

Lysine succinylation, one of post-translational acylations conserved from eukaryotes to bacteria, plays regulatory roles in various cellular processes. However, much remains unknown about the general and specific characteristics of lysine succinylation among bacteria, and about its functions different from those of other acylations. In this study, we characterized lysine succinylation, a newly discovered widespread type of lysine acylation in five bacterial species with different characteristics such as optimal growth temperature and cell wall structure. This study is the first to demonstrate that succinylation is general phenomenon occurring not only in mesophiles but also in thermophiles. Mapping of succinylation sites on protein structures revealed that succinylation occurs at many lysine residues important for protein function. Comparison of the succinylation sites in the five bacterial species provides insights regarding common protein regulation mechanisms utilizing lysine succinylation. Many succinylation sites were conserved among five bacteria, especially between Geobacillus kaustophilus and Bacillus subtilis, some of which are functionally important sites. Furthermore, systematic comparison of the succinyl-proteome results and our previous propionyl-proteome results showed that the abundance of these two types of acylations is considerably different among the five bacteria investigated. Many succinylation and propionylation events were detected in G. kaustophilus, whereas Escherichia coli and B. subtilis exhibited high succinylation and low propionylation; low succinylation and high propionylation were identified in Thermus thermophilus, and low succinylation and propionylation were observed in Rhodothermus marinus. Comparison of the characteristics of lysine succinylation and lysine propionylation suggested these two types of acylation play different roles in cellular processes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Adenosine kinase is a potential target for development of new types of drugs. The COG1839 family has been defined as "adenosine-specific kinase" family based on structural analysis and the adenosine-binding ability of a family member, PAE2307. However, there has been no experimental evidence with regard to the enzymatic function of this protein family. Here we measured the enzymatic activity of TTHA1091, a COG1839 family protein from Thermus thermophilus HB8. The phosphorylation of adenosine by TTHA1091 was undetectable when ATP or ADP were used as phosphate donor. However, the degradation of ADP to AMP was detected, indicating that this protein possessed adenosine diphosphatase (ADPase) activity. The (ADPase) activity was inhibited by divalent cations and was specific to ADP and CDP. Thus, this study provides the first experimental evidence for the enzymatic function of the "adenosine-specific kinase" family and suggests a need to reexamine its functional annotation.

In prokaryotic cells, genomic DNA forms an aggregated structure with various nucleoid-associated proteins (NAPs). The functions of genomic DNA are cooperatively modulated by NAPs, of which HU is considered to be one of the most important. HU binds double-stranded DNA (dsDNA) and serves as a structural modulator in the genome architecture. It plays important roles in diverse DNA functions, including replication, segregation, transcription and repair. Interestingly, it has been reported that HU also binds single-stranded DNA (ssDNA) regardless of sequence. However, structural analysis of HU with ssDNA has been lacking, and the functional relevance of this binding remains elusive. In this study, we found that ssDNA induced a significant change in the secondary structure of Thermus thermophilus HU (TtHU), as observed by analysis of circular dichroism spectra. Notably, this change in secondary structure was sequence specific, because the complementary ssDNA or dsDNA did not induce the change. Structural analysis using nuclear magnetic resonance confirmed that TtHU and this ssDNA formed a unique structure, which was different from the previously reported structure of HU in complex with dsDNA. Our data suggest that TtHU undergoes a distinct structural change when it associates with ssDNA of a specific sequence and subsequently exerts a yet-to-be-defined function.

Objectives: This study was conducted to clarify the effects of agarwood on histamine release from mast cells in rats and on the scratching behaviors in mice., Methods: Histamine release from rat mast cells induced by compound 48/80 or concanavalin A (Con A) and compound 48/80-induced scratching behavior in mice were examined to investigate the effects of agarwood. The hyaluronidase activity and the 3',5'-cyclic adenosine monophosphate (cAMP) levels in mast cells were examined to investigate the mechanisms for the inhibition of histamine release. The correlation between the inhibitory effects of agarwood on histamine release and the content of its typical ingredients, a 2-(2-phenylethyl)chromone derivatives, was analyzed using thin-layer chromatography., Results: Agarwood showed an inhibitory effect on mast-cell histamine release induced by compound 48/80 or Con A without any effect on hyaluronidase activity; this effect involves an increase in the cAMP levels in mast cells. Oral administration of agarwood showed an inhibitory effect on compound 48/80-induced scratching behavior in mice. The inhibitory effects of agarwood on histamine release were quite different, depending on the area where the agarwood was produced, its quality, and its market price. No correlation was found between the inhibitory effects of agarwood on histamine release and the typical ingredients of agarwood, which are 2-(2-phenylethyl)chromone derivatives., Conclusion: These results show that agarwood inhibits histamine release from mast cells partially through an increase in the cAMP levels in cells. We suggest that some active ingredients of agarwood must be effective on oral intake and that agarwood can be used to treat patients with a number of conditions, including urticaria, atopic dermatitis, and bronchial asthma, in which an increase in histamine release occurs. Differences in the pharmacological effects of this crude drug among markets may provide important information for the quality control of this herbal medicine., Competing Interests: The authors declare that there are no conflict of interest.

The crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii were determined and their structural characteristics were analyzed. For PurS from T. thermophilus, two structures were determined using two crystals that were grown in different conditions. The four structures in the dimeric form were almost identical to one another despite their relatively low sequence identities. This is also true for all PurS structures determined to date. A few residues were conserved among PurSs and these are located at the interaction site with PurL and PurQ, the other subunits of the formylglycinamide ribonucleotide amidotransferase. Molecular-dynamics simulations of the PurS dimer as well as a model of the complex of the PurS dimer, PurL and PurQ suggest that PurS plays some role in the catalysis of the enzyme by its bending motion.