pH-dependent regulation of an acidophilic O-acetylhomoserine sulfhydrylase from Lactobacillus plantarum.

Bacteria have two routes for the l-methionine biosynthesis. In one route called the direct sulfuration pathway, acetylated l-homoserine is directly converted into l-homocysteine. The reaction using H2S as the second substrate is catalyzed by a pyridoxal 5'-phosphate-dependent enzyme, O-acetylhomoserine sulfhydrylase (OAHS). In the present study, we determined the enzymatic functions and the structures of OAHS from Lactobacillus plantarum (LpOAHS). The LpOAHS enzyme exhibited the highest catalytic activity under the weak acidic pH condition. In addition, crystallographic analysis revealed that the enzyme takes two distinct structures, open and closed forms. In the closed form, two acidic residues are sterically clustered. The proximity may cause the electrostatic repulsion, inhibiting the formation of the closed form under the neutral to the basic pH conditions. We concluded that the pH-dependent regulation mechanism using the two acidic residues contributes to the acidophilic feature of the enzyme. IMPORTANCE: In the present study, we can elucidate the pH-dependent regulation mechanism of the acidophilic OAHS. The acidophilic feature of the enzyme is caused by the introduction of an acidic residue to the neighborhood of the key acidic residue acting as a switch for the structural interconversion. The strategy may be useful in the field of protein engineering to change the optimal pH of the enzymes. In addition, this study may be useful for the development of antibacterial drugs because the l-methionine synthesis essential for bacteria is inhibited by the OAHS inhibitors. The compounds that can inhibit the interconversion between the open and closed forms of OAHS may become antibacterial drugs.

Lectins engineered to favor a glycan-binding conformation have enhanced antiviral activity.

Homologues of the Oscillatoria agardhii agglutinin (OAA) lectins contain a sequence repeat of ∼66 amino acids, with the number of tandem repeats varying across family members. OAA homologues bind high-mannose glycans on viral surface proteins, thereby interfering with viral entry into host cells. As such, OAA homologues have potential utility as antiviral agents, but a more detailed understanding of their structure-function relationships would enable us to develop improved constructs. Here, we determined the X-ray crystal structure of free and glycan-bound forms of Pseudomonas taiwanensis lectin (PTL), an OAA-family lectin consisting of two tandem repeats. Like other OAA-family lectins, PTL exhibited a ß-barrel-like structure with two symmetrically positioned glycan-binding sites at the opposite ends of the barrel. Upon glycan binding, the conformation of PTL undergoes a more significant change than expected from previous OAA structural analysis. Moreover, the electron density of the bound glycans suggested that the binding affinities are different at the two binding sites. Next, based on analysis of these structures, we used site-specific mutagenesis to create PTL constructs expected to increase the population with a conformation suitable for glycan binding. The engineered PTLs were examined for their antiviral activity against the influenza virus. Interestingly, some exhibited stronger activity compared with that of the parent PTL. We propose that our approach is effective for the generation of potential microbicides with enhanced antiviral activity.

Crystal structure of O-ureidoserine racemase found in the d-cycloserine biosynthetic pathway.

The O-ureidoserine racemase (DcsC) is an enzyme found from the biosynthetic gene cluster of antitubercular agent d-cycloserine. Although DcsC is homologous to diaminopimelate epimerase (DapF) that catalyzes the interconversion between ll- and dl-diaminopimelic acid, it specifically catalyzes the interconversion between O-ureido-l-serine and its enantiomer. Here we determined the crystal structure of DcsC at a resolution of 2.12 Å, implicating that the catalytic mechanism of DcsC shares similarity with that of DapF. Comparing the structure of the active center of DcsC to that of DapF, Thr72, Thr198, and Tyr219 of DcsC are likely to be involved in the substrate specificity.

Structural Insight into the Interaction of Sendai Virus C Protein with Alix To Stimulate Viral Budding.

Sendai virus (SeV), belonging to the Respirovirus genus of the family Paramyxoviridae, harbors an accessory protein, named C protein, which facilitates viral pathogenicity in mice. In addition, the C protein is known to stimulate the budding of virus-like particles by binding to the host ALG-2 interacting protein X (Alix), a component of the endosomal sorting complexes required for transport (ESCRT) machinery. However, small interfering RNA (siRNA)-mediated gene knockdown studies suggested that neither Alix nor C protein is related to SeV budding. In the present study, we determined the crystal structure of a complex comprising the C-terminal half of the C protein (Y3) and the Bro1 domain of Alix at a resolution of 2.2 Å to investigate the role of the complex in SeV budding. The structure revealed that a novel consensus sequence, LXXW, which is conserved among Respirovirus C proteins, is important for Alix binding. SeV possessing a mutated C protein with reduced Alix-binding affinity showed impaired virus production, which correlated with the binding affinity. Infectivity analysis showed a 160-fold reduction at 12 h postinfection compared with nonmutated virus, while C protein competes with CHMP4, one subunit of the ESCRT-III complex, for binding to Alix. All together, these results highlight the critical role of C protein in SeV budding. IMPORTANCE Human parainfluenza virus type I (hPIV1) is a respiratory pathogen affecting young children, immunocompromised patients, and the elderly, with no available vaccines or antiviral drugs. Sendai virus (SeV), a murine counterpart of hPIV1, has been studied extensively to determine the molecular and biological properties of hPIV1. These viruses possess a multifunctional accessory protein, C protein, which is essential for stimulating viral reproduction, but its role in budding remains controversial. In the present study, the crystal structure of the C-terminal half of the SeV C protein associated with the Bro1 domain of Alix, a component of cell membrane modulating machinery ESCRT, was elucidated. Based on the structure, we designed mutant C proteins with different binding affinities to Alix and showed that the interaction between C and Alix is vital for viral budding. These findings provide new insights into the development of new antiviral drugs against hPIV1.

Structural analysis of the STAT1:STAT2 heterodimer revealed the mechanism of Sendai virus C protein-mediated blockade of type 1 interferon signaling.

Sendai virus (SeV), which causes respiratory diseases in rodents, possesses the C protein that blocks the signal transduction of interferon (IFN), thereby escaping from host innate immunity. We previously demonstrated by using protein crystallography that two molecules of Y3 (the C-terminal half of the C protein) can bind to the homodimer of the N-terminal domain of STAT1 (STAT1ND), elucidating the mechanism of inhibition of IFN-γ signal transduction. SeV C protein also blocks the signal transduction of IFN-α/ß by inhibiting the phosphorylation of STAT1 and STAT2, although the mechanism for the inhibition is unclear. Therefore, we sought to elucidate the mechanism of inhibition of the IFN signal transduction via STAT1 and STAT2. Small angle X-ray scattering analysis indicated that STAT1ND associates with the N-terminal domain of STAT2 (STAT2ND) with the help of a Gly-rich linker. We generated a linker-less recombinant protein possessing a STAT1ND:STAT2ND heterodimeric structure via an artificial disulfide bond. Analytical size-exclusion chromatography and surface plasmon resonance revealed that one molecule of Y3 can associate with a linker-less recombinant protein. We propose that one molecule of C protein associates with the STAT1:STAT2 heterodimer, inducing a conformational change to an antiparallel form, which is easily dephosphorylated. This suggests that association of C protein with the STAT1ND:STAT2ND heterodimer is an important factor to block the IFN-α/ß signal transduction.

Structural Basis of the Inhibition of STAT1 Activity by Sendai Virus C Protein.

UNLABELLED: Sendai virus (SeV) C protein inhibits the signal transduction pathways of interferon alpha/beta (IFN-α/ß) and IFN-γ by binding to the N-terminal domain of STAT1 (STAT1ND), thereby allowing SeV to escape from host innate immunity. Here we determined the crystal structure of STAT1ND associated with the C-terminal half of the C protein (Y3 [amino acids 99 to 204]) at a resolution of 2.0 Å. This showed that two molecules of Y3 symmetrically bind to each niche created between two molecules of the STAT1ND dimer. Molecular modeling suggested that an antiparallel form of the full-length STAT1 dimer can bind only one Y3 molecule and that a parallel form can bind two Y3 molecules. Affinity analysis demonstrated anticooperative binding of two Y3 molecules with the STAT1 dimer, which is consistent with the hypothetical model that the second Y3 molecule can only target the STAT1 dimer in a parallel form. STAT1 with excess amounts of Y3 was prone to inhibit the dephosphorylation at Tyr(701) by a phosphatase. In an electrophoretic mobility shift assay, tyrosine-phosphorylated STAT1 (pY-STAT1) with Y3 associated with the γ-activated sequence, probably as high-molecular-weight complexes (HMWCs), which may account for partial inhibition of a reporter assay from IFN-γ by Y3. Our study suggests that the full-length C protein interferes with the domain arrangement of the STAT1 dimer, leading to the accumulation of pY-STAT1 and the formation of HMWCs. In addition, we discuss the mechanism by which phosphorylation of STAT2 is inhibited in the presence of the C protein after stimulation by IFN-α/ß. IMPORTANCE: Sendai virus, a paramyxovirus that causes respiratory diseases in rodents, possesses the C protein, which inhibits the signal transduction pathways of interferon alpha/beta (IFN-α/ß) and IFN-γ by binding to the transcription factor STAT1. In virus-infected cells, phosphorylation of STAT1 at the Tyr(701) residue is potently enhanced, although transcription by STAT1 is inert. Here, we determined the crystal structure of the N-terminal domain of STAT1 associated with the C-terminal half of the C protein. Molecular modeling and experiments suggested that the two C proteins bind to and stabilize the parallel form of the STAT1 dimer, which are likely to be phosphorylated at Tyr(701), further inducing high-molecular-weight complex formation and inhibition of transcription by IFN-γ. We also discuss the possible mechanism of inhibition of the IFN-α/ß pathways by the C protein. This is the first structural report of the C protein, suggesting a mechanism of evasion of the paramyxovirus from innate immunity.

Human ß-Defensin 3 Inhibition of P. gingivalis LPS-Induced IL-1ß Production by BV-2 Microglia through Suppression of Cathepsins B and L.

Cathepsin B (CatB) is thought to be essential for the induction of Porphyromonas gingivalis lipopolysaccharide (Pg LPS)-induced Alzheimer's disease-like pathologies in mice, including interleukin-1ß (IL-1ß) production and cognitive decline. However, little is known about the role of CatB in Pg virulence factor-induced IL-1ß production by microglia. We first subjected IL-1ß-luciferase reporter BV-2 microglia to inhibitors of Toll-like receptors (TLRs), IκB kinase, and the NLRP3 inflammasome following stimulation with Pg LPS and outer membrane vesicles (OMVs). To clarify the involvement of CatB, we used several known CatB inhibitors, including CA-074Me, ZRLR, and human ß-defensin 3 (hBD3). IL-1ß production in BV-2 microglia induced by Pg LPS and OMVs was significantly inhibited by the TLR2 inhibitor C29 and the IκB kinase inhibitor wedelolactonne, but not by the NLRPs inhibitor MCC950. Both hBD3 and CA-074Me significantly inhibited Pg LPS-induced IL-1ß production in BV-2 microglia. Although CA-074Me also suppressed OMV-induced IL-1ß production, hBD3 did not inhibit it. Furthermore, both hBD3 and CA-074Me significantly blocked Pg LPS-induced nuclear NF-κB p65 translocation and IκBα degradation. In contrast, hBD3 and CA-074Me did not block OMV-induced nuclear NF-κB p65 translocation or IκBα degradation. Furthermore, neither ZRLR, a specific CatB inhibitor, nor shRNA-mediated knockdown of CatB expression had any effect on Pg virulence factor-induced IL-1ß production. Interestingly, phagocytosis of OMVs by BV-2 microglia induced IL-1ß production. Finally, the structural models generated by AlphaFold indicated that hBD3 can bind to the substrate-binding pocket of CatB, and possibly CatL as well. These results suggest that Pg LPS induces CatB/CatL-dependent synthesis and processing of pro-IL-1ß without activation of the NLRP3 inflammasome. In contrast, OMVs promote the synthesis and processing of pro-IL-1ß through CatB/CatL-independent phagocytic mechanisms. Thus, hBD3 can improve the IL-1ß-associated vicious inflammatory cycle induced by microglia through inhibition of CatB/CatL.

Crystallographic study to determine the substrate specificity of an L-serine-acetylating enzyme found in the D-cycloserine biosynthetic pathway.

DcsE, one of the enzymes found in the d-cycloserine biosynthetic pathway, displays a high sequence homology to l-homoserine O-acetyltransferase (HAT), but it prefers l-serine over l-homoserine as the substrate. To clarify the substrate specificity, in the present study we determined the crystal structure of DcsE at a 1.81-Å resolution, showing that the overall structure of DcsE is similar to that of HAT, whereas a turn region to form an oxyanion hole is obviously different between DcsE and HAT: in detail, the first and last residues in the turn of DcsE are Gly(52) and Pro(55), respectively, but those of HAT are Ala and Gly, respectively. In addition, more water molecules were laid on one side of the turn region of DcsE than on that of HAT, and a robust hydrogen-bonding network was formed only in DcsE. We created a HAT-like mutant of DcsE in which Gly(52) and Pro(55) were replaced by Ala and Gly, respectively, showing that the mutant acetylates l-homoserine but scarcely acetylates l-serine. The crystal structure of the mutant DcsE shows that the active site, including the turn and its surrounding waters, is similar to that of HAT. These findings suggest that a methyl group of the first residue in the turn of HAT plays a role in excluding the binding of l-serine to the substrate-binding pocket. In contrast, the side chain of the last residue in the turn of DcsE may need to form an extensive hydrogen-bonding network on the turn, which interferes with the binding of l-homoserine.

Establishment of an in vitro D-cycloserine-synthesizing system by using O-ureido-L-serine synthase and D-cycloserine synthetase found in the biosynthetic pathway.

We have recently cloned a DNA fragment containing a gene cluster that is responsible for the biosynthesis of an antituberculosis antibiotic, D-cycloserine. The gene cluster is composed of 10 open reading frames, designated dcsA to dcsJ. Judging from the sequence similarity between each putative gene product and known proteins, DcsC, which displays high homology to diaminopimelate epimerase, may catalyze the racemization of O-ureidoserine. DcsD is similar to O-acetylserine sulfhydrylase, which generates L-cysteine using O-acetyl-L-serine with sulfide, and therefore, DcsD may be a synthase to generate O-ureido-L-serine using O-acetyl-L-serine and hydroxyurea. DcsG, which exhibits similarity to a family of enzymes with an ATP-grasp fold, may be an ATP-dependent synthetase converting O-ureido-D-serine into D-cycloserine. In the present study, to characterize the enzymatic functions of DcsC, DcsD, and DcsG, each protein was overexpressed in Escherichia coli and purified to near homogeneity. The biochemical function of each of the reactions catalyzed by these three proteins was verified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and, in some cases, mass spectrometry. The results from this study demonstrate that by using a mixture of the three purified enzymes and the two commercially available substrates O-acetyl-L-serine and hydroxyurea, synthesis of D-cycloserine was successfully attained. These in vitro studies yield the conclusion that DcsD and DcsG are necessary for the syntheses of O-ureido-L-serine and D-cycloserine, respectively. DcsD was also able to catalyze the synthesis of L-cysteine when sulfide was added instead of hydroxyurea. Furthermore, the present study shows that DcsG can also form other cyclic d-amino acid analogs, such as D-homocysteine thiolactone.

Virus purification highlights the high susceptibility of SARS-CoV-2 to a chlorine-based disinfectant, chlorous acid.

Chlorous acid water (HClO2) is known for its antimicrobial activity. In this study, we attempted to accurately assess the ability of chlorous acid water to inactivate SARS-CoV-2. When using cell culture supernatants of infected cells as the test virus, the 99% inactivation concentration (IC99) for the SARS-CoV-2 D614G variant, as well as the Delta and Omicron variants, was approximately 10ppm of free chlorine concentration with a reaction time of 10 minutes. On the other hand, in experiments using a more purified virus, the IC99 of chlorous acid water was 0.41-0.74ppm with a reaction time of 1 minute, showing a strong inactivation capacity over 200 times. With sodium hypochlorite water, the IC99 was 0.54ppm, confirming that these chlorine compounds have a potent inactivation effect against SARS-CoV-2. However, it became clear that when using cell culture supernatants of infected cells as the test virus, the effect is masked by impurities such as amino acids contained therein. Also, when proteins (0.5% polypeptone, or 0.3% BSA + 0.3% sheep red blood cells, or 5% FBS) were added to the purified virus, the IC99 values became high, ranging from 5.3 to 76ppm with a reaction time of 10 minutes, significantly reducing the effect. However, considering that the usual usage concentration is 200ppm, it was shown that chlorous acid water can still exert sufficient disinfection effects even in the presence of proteins. Further research is needed to confirm the practical applications and effects of chlorous acid water, but it has the potential to be an important tool for preventing the spread of SARS-CoV-2.

Optineurin deficiency impairs autophagy to cause interferon beta overproduction and increased survival of mice following viral infection.

BACKGROUND: Optineurin (OPTN) is associated with several human diseases, including amyotrophic lateral sclerosis (ALS), and is involved in various cellular processes, including autophagy. Optineurin regulates the expression of interferon beta (IFNß), which plays a central role in the innate immune response to viral infection. However, the role of optineurin in response to viral infection has not been fully clarified. It is known that optineurin-deficient cells produce more IFNß than wild-type cells following viral infection. In this study, we investigate the reasons for, and effects of, IFNß overproduction during optineurin deficiency both in vitro and in vivo. METHODS: To investigate the mechanism of IFNß overproduction, viral nucleic acids in infected cells were quantified by RT-qPCR and the autophagic activity of optineurin-deficient cells was determined to understand the basis for the intracellular accumulation of viral nucleic acids. Moreover, viral infection experiments using optineurin-disrupted (Optn-KO) animals were performed with several viruses. RESULTS: IFNß overproduction following viral infection was observed not only in several types of optineurin-deficient cell lines but also in Optn-KO mice and human ALS patient cells carrying mutations in OPTN. IFNß overproduction in Optn-KO cells was revealed to be caused by excessive accumulation of viral nucleic acids, which was a consequence of reduced autophagic activity caused by the loss of optineurin. Additionally, IFNß overproduction in Optn-KO mice suppressed viral proliferation, resulting in increased mouse survival following viral challenge. CONCLUSION: Our findings indicate that the combination of optineurin deficiency and viral infection leads to IFNß overproduction in vitro and in vivo. The effects of optineurin deficiency are elicited by viral infection, therefore, viral infection may be implicated in the development of optineurin-related diseases.

A molecular mechanism for copper transportation to tyrosinase that is assisted by a metallochaperone, caddie protein.

The Cu(II)-soaked crystal structure of tyrosinase that is present in a complex with a protein, designated "caddie," which we previously determined, possesses two copper ions at its catalytic center. We had identified two copper-binding sites in the caddie protein and speculated that copper bound to caddie may be transported to the tyrosinase catalytic center. In our present study, at a 1.16-1.58 Å resolution, we determined the crystal structures of tyrosinase complexed with caddie prepared by altering the soaking time of the copper ion and the structures of tyrosinase complexed with different caddie mutants that display little or no capacity to activate tyrosinase. Based on these structures, we propose a molecular mechanism by which two copper ions are transported to the tyrosinase catalytic center with the assistance of caddie acting as a metallochaperone.

Heme protein and hydroxyarginase necessary for biosynthesis of D-cycloserine.

We have recently cloned a D-cycloserine (DCS) biosynthetic gene cluster that consists of 10 genes, designated dcsA~dcsJ, from Streptomyces lavendulae ATCC 11924 (16). In the predicted pathway of hydroxyurea (HU) formation in DCS biosynthesis, L-arginine (L-Arg) must first be hydroxylated, prior to the hydrolysis of N(ω)-hydroxy-L-arginine (NHA) by DcsB, an arginase homolog. The hydroxylation of L-Arg is known to be catalyzed by nitric oxide synthase (NOS). In this study, to verify the supply route of HU, we created a dcsB-disrupted mutant, ΔdcsB. While the mutant lost DCS productivity, its productivity was restored by complementation of dcsB, and also by the addition of HU but not NHA, suggesting that HU is supplied by DcsB. A NOS-encoding gene, nos, from S. lavendulae chromosome was cloned, to create a nos-disrupted mutant. However, the mutant maintained the DCS productivity, suggesting that NOS is not necessary for DCS biosynthesis. To clarify the identity of an enzyme necessary for NHA formation, a dcsA-disrupted mutant, designated ΔdcsA, was also created. The mutant lost DCS productivity, whereas the DCS productivity was restored by complementation of dcsA. The addition of NHA to the culture medium of ΔdcsA mutant was also effective to restore DCS production. These results indicate that the dcsA gene product, DcsA, is an enzyme essential to generate NHA as a precursor in the DCS biosynthetic pathway. Spectroscopic analyses of the recombinant DcsA revealed that it is a heme protein, supporting an idea that DcsA is an enzyme catalyzing hydroxylation.

Catalytic mechanism of DcsB: Arginase framework used for hydrolyzing its inhibitor.

DcsB, an enzyme produced from the d-cycloserine biosynthetic gene cluster, displays moderate similarity to arginase in the sequence and three-dimensional structure. Arginase is a ubiquitous enzyme hydrolyzing l-arginine to generate l-ornithine and urea, whereas DcsB hydrolyzes Nω -hydroxy-l-arginine (l-NOHA), an arginase inhibitor, to generate l-ornithine and hydroxyurea. We determined the crystal structure of DcsB associated with l-ornithine and that with the tetrahedral derivative of 2(S)-amino-6-boronohexanoic acid, whose boron atom forms a covalent bond with an oxygen atom bridging two manganese ions at the active center. The substrate-binding pocket of DcsB is narrower than that of arginase, suggesting that DcsB is unsuitable for the binding of l-NOHA in an inhibitory manner. The transition state-like structure demonstrated that Asp210 and Glu241 have a role to trap a positively charged ion near the dimanganese cluster. Kinetic analysis using the mutated DcsB showed that the enzyme employs different catalytic mechanisms under the neutral and alkaline pH conditions. Glu241 in DcsB is likely involved in the recognition of the hydroxyguanidino group of l-NOHA, whereas Asp210, in cooperation with Glu241, seems to contribute to the reactivity toward the protonated l-NOHA, which is a preferable species under the neutral pH conditions. After entering of the protonated l-NOHA to the substrate-binding pocket of DcsB, a hydronium ion may be trapped at the positive ion-binding site. Then, the ion serves as a specific acid catalyst to facilitate the collapse of the tetrahedral intermediate of l-NOHA.

Catalytic mechanism of bleomycin N-acetyltransferase proposed on the basis of its crystal structure.

Bleomycin (Bm) N-acetyltransferase, BAT, is a self-resistance determinant in Bm-producing Streptomyces verticillus ATCC15003. In our present study, we crystallized BAT under both a terrestrial and a microgravity environment in the International Space Station. In addition to substrate-free BAT, the crystal structures of BAT in a binary complex with CoA and in a ternary complex with Bm and CoA were determined. BAT forms a dimer structure via interaction of its C-terminal domains in the monomers. However, each N-terminal domain in the dimer is positioned without mutual interaction. The tunnel observed in the N-terminal domain of BAT has two entrances: one that adopts a wide funnel-like structure necessary to accommodate the metal-binding domain of Bm, and another narrow entrance that accommodates acetyl-CoA (AcCoA). A groove formed on the dimer interface of two BAT C-terminal domains accommodates the DNA-binding domain of Bm. In a ternary complex of BAT, BmA(2), and CoA, a thiol group of CoA is positioned near the primary amine of Bm at the midpoint of the tunnel. This proximity ensures efficient transfer of an acetyl group from AcCoA to the primary amine of Bm. Based on the BAT crystal structure and the enzymatic kinetic study, we propose that the catalytic mode of BAT takes an ordered-like mechanism.

The basicity of an active-site water molecule discriminates between tyrosinase and catechol oxidase activity.

Tyrosinase (Ty) and catechol oxidase (CO) are members of type-3 copper enzymes. While Ty catalyzes both phenolase and catecholase reactions, CO catalyzes only the latter reaction. In the present study, Ty was found to catalyze the catecholase reaction, but hardly the phenolase reaction in the presence of the metallochaperon called "caddie protein (Cad)". The ability of the substrates to dissociate the motif shielding the active-site pocket seems to contribute critically to the substrate specificity of Ty. In addition, a mutation at the N191 residue, which forms a hydrogen bond with a water molecule near the active center, decreased the inherent ratio of phenolase versus catecholase activity. Unlike the wild-type complex, reaction intermediates were not observed when the catalytic reaction toward the Y98 residue of Cad was progressed in the crystalline state. The increased basicity of the water molecule may be necessary to inhibit the proton transfer from the conjugate acid to a hydroxide ion bridging the two copper ions. The deprotonation of the substrate hydroxyl by the bridging hydroxide seems to be significant for the efficient catalytic cycle of the phenolase reaction.

Ethanol Susceptibility of SARS-CoV-2 and Other Enveloped Viruses.

Ethanol is an effective disinfectant against the novel coronavirus SARS-CoV-2. However, its effective concentration has not been shown, and we therefore analyzed the effects of different concentrations of ethanol on SARS-CoV-2. When SARS-CoV-2 was treated with varying ethanol concentrations and examined for changes in infectivity, the ethanol concentration at which 99% of the infectious titers were reduced was 24.1% (w/w) [29.3% (v/v)]. For reference, ethanol susceptibility was also examined with other envelope viruses, including influenza virus, vesicular stomatitis virus in the family Rhabdoviridae, and Newcastle disease virus in the family Paramyxoviridae, and the 99% inhibitory concentrations were found to be 28.8%(w/w) [34.8% (v/v)], 24.0% (w/w) [29.2% (v/v)], and 13.3% (w/w) [16.4% (v/v)], respectively. Some differences from SARS-CoV-2 were observed, but the differences were not significant. It was concluded that ethanol at a concentration of 30%(w/w) [36.2% (v/v)] almost completely inactivates SARS-CoV-2.

Molecular cloning and heterologous expression of a biosynthetic gene cluster for the antitubercular agent D-cycloserine produced by Streptomyces lavendulae.

In the present study, we successfully cloned a 21-kb DNA fragment containing a d-cycloserine (DCS) biosynthetic gene cluster from a DCS-producing Streptomyces lavendulae strain, ATCC 11924. The putative gene cluster consists of 10 open reading frames (ORFs), designated dcsA to dcsJ. This cluster includes two ORFs encoding D-alanyl-D-alanine ligase (dcsI) and a putative membrane protein (dcsJ) as the self-resistance determinants of the producer organism, indicated by our previous work. When the 10 ORFs were introduced into DCS-nonproducing Streptomyces lividans 66 as a heterologous host cell, the transformant acquired DCS productivity. This reveals that the introduced genes are responsible for the biosynthesis of DCS. As anticipated, the disruption of dcsG, seen in the DCS biosynthetic gene cluster, made it possible for the strain ATCC 11924 to lose its DCS production. We here propose the DCS biosynthetic pathway. First, L-serine is O acetylated by a dcsE-encoded enzyme homologous to homoserine O-acetyltransferase. Second, O-acetyl-L-serine accepts hydroxyurea via an O-acetylserine sulfhydrylase homolog (dcsD product) and forms O-ureido-L-serine. The hydroxyurea must be supplied by the catalysis of a dcsB-encoded arginase homolog using the L-arginine derivative, N(G)-hydroxy-L-arginine. The resulting O-ureido-L-serine is then racemized to O-ureido-D-serine by a homolog of diaminopimelate epimerase. Finally, O-ureido-D-serine is cyclized to form DCS with the release of ammonia and carbon dioxide. The cyclization must be done by the dcsG or dcsH product, which belongs to the ATP-grasp fold family of protein.

Crystal structure of an Nω-hydroxy-L-arginine hydrolase found in the D-cycloserine biosynthetic pathway.

DcsB, one of the enzymes encoded in the D-cycloserine (D-CS) biosynthetic gene cluster, displays a high sequence homology to arginase, which contains two manganese ions in the active site. However, DcsB hydrolyzes Nω-hydroxy-L-arginine, but not L-arginine, to supply hydroxyurea for the biosynthesis of D-CS. Here, the crystal structure of DcsB was determined at a resolution of 1.5 Šusing anomalous scattering from the manganese ions. In the crystal structure, DscB generates an artificial dimer created by the open and closed forms. Gel-filtration analysis demonstrated that DcsB is a monomeric protein, unlike arginase, which forms a trimeric structure. The active center containing the binuclear manganese cluster differs between DcsB and arginase. In DcsB, one of the ligands of the MnA ion is a cysteine, while the corresponding residue in arginase is a histidine. In addition, DcsB has no counterpart to the histidine residue that acts as a general acid/base during the catalytic reaction of arginase. The present study demonstrates that DcsB has a unique active site that differs from that of arginase.