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1.
Appl Environ Microbiol ; 90(5): e0011824, 2024 May 21.
Article in English | MEDLINE | ID: mdl-38568076

ABSTRACT

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.


Subject(s)
Bacterial Proteins , Lactobacillus plantarum , Lactobacillus plantarum/enzymology , Lactobacillus plantarum/genetics , Lactobacillus plantarum/metabolism , Hydrogen-Ion Concentration , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Carbon-Oxygen Lyases
2.
J Biol Chem ; 296: 100698, 2021.
Article in English | MEDLINE | ID: mdl-33895142

ABSTRACT

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.


Subject(s)
Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Lectins/metabolism , Lectins/pharmacology , Polysaccharides/metabolism , Protein Engineering , Antiviral Agents/chemistry , Crystallography, X-Ray , Lectins/chemistry , Lectins/genetics , Models, Molecular , Orthomyxoviridae/drug effects , Protein Binding , Protein Conformation, beta-Strand
3.
Proteins ; 90(4): 912-918, 2022 04.
Article in English | MEDLINE | ID: mdl-34877716

ABSTRACT

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.


Subject(s)
Cycloserine , Racemases and Epimerases , Biosynthetic Pathways , Crystallography, X-Ray , Cycloserine/chemistry , Cycloserine/metabolism , Multigene Family , Racemases and Epimerases/genetics , Racemases and Epimerases/metabolism , Serine/metabolism
4.
J Virol ; 95(19): e0081521, 2021 09 09.
Article in English | MEDLINE | ID: mdl-34287046

ABSTRACT

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.


Subject(s)
Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/chemistry , Endosomal Sorting Complexes Required for Transport/metabolism , Sendai virus/physiology , Viral Proteins/chemistry , Viral Proteins/metabolism , Virus Release , Amino Acid Sequence , Animals , Binding, Competitive , Cell Line , Crystallography, X-Ray , Humans , Interferon-alpha/genetics , Interferon-alpha/metabolism , Interferon-beta/genetics , Interferon-beta/metabolism , Models, Molecular , Protein Binding , Protein Conformation , Protein Domains , Sendai virus/chemistry , Sendai virus/genetics , Sendai virus/metabolism , Signal Transduction , Virion/physiology
5.
PLoS Biol ; 16(12): e3000077, 2018 12.
Article in English | MEDLINE | ID: mdl-30596633

ABSTRACT

Tyrosinase (EC 1.14.18.1), a copper-containing monooxygenase, catalyzes the conversion of phenol to the corresponding ortho-quinone. The Streptomyces tyrosinase is generated as a complex with a "caddie" protein that facilitates the transport of two copper ions into the active center. In our previous study, the Tyr98 residue in the caddie protein, which is accommodated in the pocket of active center of tyrosinase, has been found to be converted to a reactive quinone through the formations of the µ-η2:η2-peroxo-dicopper(II) and Cu(II)-dopasemiquinone intermediates. Until now-despite extensive studies for the tyrosinase reaction based on the crystallographic analysis, low-molecular-weight models, and computer simulations-the catalytic mechanism has been unable to be made clear at an atomic level. To make the catalytic mechanism of tyrosinase clear, in the present study, the cryo-trapped crystal structures were determined at very high resolutions (1.16-1.70 Å). The structures suggest the existence of an important step for the tyrosinase reaction that has not yet been found: that is, the hydroxylation reaction is triggered by the movement of CuA, which induces the syn-to-anti rearrangement of the copper ligands after the formation of µ-η2:η2-peroxo-dicopper(II) core. By the rearrangement, the hydroxyl group of the substrate is placed in an equatorial position, allowing the electrophilic attack to the aromatic ring by the Cu2O2 oxidant.


Subject(s)
Copper/metabolism , Monophenol Monooxygenase/physiology , Monophenol Monooxygenase/ultrastructure , Benzoquinones/metabolism , Binding Sites/physiology , Catalysis , Crystallography, X-Ray/methods , Hydroxylation , Ligands , Models, Molecular , Monophenol Monooxygenase/metabolism , Phenols/chemistry , Streptomyces/genetics , Streptomyces/metabolism , Tyrosine/metabolism
6.
J Biol Chem ; 292(48): 19752-19766, 2017 12 01.
Article in English | MEDLINE | ID: mdl-28978648

ABSTRACT

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.


Subject(s)
Interferon Type I/metabolism , STAT1 Transcription Factor/metabolism , STAT2 Transcription Factor/metabolism , Sendai virus/metabolism , Signal Transduction , Viral Proteins/metabolism , Cell Line , Crystallography, X-Ray , Dimerization , Humans , Phosphorylation , Protein Conformation , STAT1 Transcription Factor/chemistry , STAT2 Transcription Factor/chemistry
7.
Appl Environ Microbiol ; 84(7)2018 04 01.
Article in English | MEDLINE | ID: mdl-29352085

ABSTRACT

We have previously shown that the lactic acid bacterium Lactobacillus brevis 174A, isolated from Citrus iyo fruit, produces a bacteriocin designated brevicin 174A, which is comprised of two antibacterial polypeptides (designated brevicins 174A-ß and 174A-γ). We have also found a gene cluster, composed of eight open reading frames (ORFs), that contains genes for the biosynthesis of brevicin 174A, self-resistance to its own bacteriocin, and two transcriptional regulatory proteins. Some lactic acid bacterial strains have a system to start the production of bacteriocin at an adequate stage of growth. Generally, the system consists of a membrane-bound histidine protein kinase (HPK) that senses a specific environmental stimulus and a corresponding response regulator (RR) that mediates the cellular response. We have previously shown that although the HPK- and RR-encoding genes are not found on the brevicin 174A biosynthetic gene cluster in the 174A strain, two putative regulatory genes, designated breD and breG, are in the gene cluster. In the present study, we demonstrate that the expression of brevicin 174A production and self-resistance is positively controlled by two transcriptional regulatory proteins, designated BreD and BreG. BreD is expressed together with BreE as the self-resistance determinant of L. brevis 174A. DNase I footprinting analysis and a promoter assay demonstrated that BreD binds to the breED promoter as a positive autoregulator. The present study also demonstrates that BreG, carrying a transmembrane domain, binds to the common promoter of breB and breC, encoding brevicins 174A-ß and 174A-γ, respectively, for positive regulation.IMPORTANCE The problem of the appearance of bacteria that are resistant to practical antibiotics and the increasing demand for safe foods have increased interest in replacing conventional antibiotics with bacteriocin produced by the lactic acid bacteria. This antibacterial substance can inhibit the growth of pathogenic bacteria without side effects on the human body. The bacteriocin that is produced by a Citrus iyo-derived Lactobacillus brevis strain inhibits the growth of pathogenic bacteria such as Listeria monocytogenes, Staphylococcus aureus, and Streptococcus mutans In general, lactic acid bacterial strains have a system to start the production of bacteriocin at an adequate stage of growth, which is called a quorum-sensing system. The system consists of a membrane-bound histidine protein kinase that senses a specific environmental stimulus and a corresponding response regulator that mediates the cellular response. The present study demonstrates that the expression of the genes encoding bacteriocin biosynthesis and the self-resistance determinant is positively controlled by two transcriptional regulatory proteins.


Subject(s)
Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Genes, Bacterial/genetics , Levilactobacillus brevis/genetics , Bacterial Proteins/metabolism , Bacteriocins/genetics , Bacteriocins/metabolism , Levilactobacillus brevis/metabolism
8.
Biochemistry ; 56(41): 5593-5603, 2017 10 17.
Article in English | MEDLINE | ID: mdl-28902505

ABSTRACT

Tyrosinase (EC 1.14.18.1), which possesses two copper ions at the active center, catalyzes a rate-limiting reaction of melanogenesis, that is, the conversion of a phenol to the corresponding ortho-quinone. The enzyme from the genus Streptomyces is generated as a complex with a "caddie" protein that assists the transport of two copper ions into the active center. In this complex, the Tyr98 residue in the caddie protein was found to be accommodated in the pocket of the active center of tyrosinase, probably in a manner similar to that of l-tyrosine as a genuine substrate of tyrosinase. Under physiological conditions, the addition of the copper ion to the complex releases tyrosinase from the complex, in accordance with the aggregation of the caddie protein. The release of the copper-bound tyrosinase was found to be accelerated by adding reducing agents under aerobic conditions. Mass spectroscopic analysis indicated that the Tyr98 residue was converted to a reactive quinone, and resonance Raman spectroscopic analysis indicated that the conversion occurred through the formations of µ-η2:η2-peroxo-dicopper(II) and Cu(II)-semiquinone. Electron paramagnetic resonance analysis under anaerobic conditions and Fourier transform infrared spectroscopic analysis using CO as a structural probe under anaerobic conditions indicated that the copper transportation process to the active center is a reversible event in the tyrosinase/caddie complex. Aggregation of the caddie protein, which is triggered by the conversion of the Tyr98 residue to dopaquinone, may ensure the generation of fully activated tyrosinase.


Subject(s)
Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Copper/metabolism , Models, Molecular , Monophenol Monooxygenase/metabolism , Streptomyces/enzymology , Amino Acid Substitution , Apoenzymes/chemistry , Apoenzymes/genetics , Apoenzymes/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Benzoquinones/chemistry , Benzoquinones/metabolism , Binding Sites , Carrier Proteins/chemistry , Carrier Proteins/genetics , Catalytic Domain , Copper/chemistry , Dihydroxyphenylalanine/analogs & derivatives , Dihydroxyphenylalanine/chemistry , Dihydroxyphenylalanine/metabolism , Enzyme Activation/drug effects , Monophenol Monooxygenase/chemistry , Monophenol Monooxygenase/genetics , Mutation , Oxidation-Reduction , Protein Aggregates/drug effects , Protein Multimerization/drug effects , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Reducing Agents/chemistry , Solubility , Tyrosine/chemistry , Tyrosine/metabolism
9.
J Virol ; 89(22): 11487-99, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26339056

ABSTRACT

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.


Subject(s)
Interferon-alpha/antagonists & inhibitors , Interferon-beta/antagonists & inhibitors , Interferon-gamma/antagonists & inhibitors , STAT1 Transcription Factor/antagonists & inhibitors , Viral Proteins/ultrastructure , Binding Sites , Cell Line , Crystallography, X-Ray , Electrophoretic Mobility Shift Assay , HEK293 Cells , Humans , Interferon-alpha/metabolism , Interferon-beta/metabolism , Models, Molecular , Phosphorylation , Protein Binding , Protein Structure, Tertiary , STAT1 Transcription Factor/metabolism , STAT1 Transcription Factor/ultrastructure , STAT2 Transcription Factor/metabolism , Sendai virus/metabolism , Signal Transduction/physiology , Viral Proteins/metabolism
10.
Appl Environ Microbiol ; 81(22): 7881-7, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26341210

ABSTRACT

Previously, we successfully cloned a d-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designated dcsA to dcsJ) from d-CS-producing Streptomyces lavendulae ATCC 11924. In this study, we put four d-CS biosynthetic genes (dcsC, dcsD, dcsE, and dcsG) in tandem under the control of the T7 promoter in an Escherichia coli host. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. When l-serine and hydroxyurea (HU), the precursors of d-CS, were incubated together with the E. coli resting cell suspension, the cells produced significant amounts of d-CS (350 ± 20 µM). To increase the productivity of d-CS, the dcsJ gene, which might be responsible for the d-CS excretion, was connected downstream of the four genes. The E. coli resting cells harboring the five genes produced d-CS at 660 ± 31 µM. The dcsD gene product, DcsD, forms O-ureido-l-serine from O-acetyl-l-serine (OAS) and HU, which are intermediates in d-CS biosynthesis. DcsD also catalyzes the formation of l-cysteine from OAS and H2S. To repress the side catalytic activity of DcsD, the E. coli chromosomal cysJ and cysK genes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the four d-CS biosynthetic genes, together with dcsJ, were incubated with l-serine and HU, the d-CS production was 980 ± 57 µM, which is comparable to that of d-CS-producing S. lavendulae ATCC 11924 (930 ± 36 µM).


Subject(s)
Anti-Infective Agents/metabolism , Cycloserine/metabolism , Escherichia coli/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Multigene Family , Organisms, Genetically Modified/genetics , Organisms, Genetically Modified/metabolism
11.
Biol Pharm Bull ; 38(12): 1902-9, 2015.
Article in English | MEDLINE | ID: mdl-26632181

ABSTRACT

In the present study, we isolated a lactic acid bacterium (LAB) from a citrus iyo fruit and identified it as Lactobacillus brevis. This plant-derived LAB strain, designated 174A, produces bacteriocin consisting of two polypeptides designated brevicin 174A-ß and 174A-γ. Although each polypeptide itself displays antibacterial activity, the ability is enhanced 100 fold by mixing both polypeptides at a 1 : 1 ratio. Significantly, brevicin 174A inhibits even the growth of several pathogenic bacteria that are more resistant to a lantibiotic bacteriocin, nisin A, which is commonly utilized as a preservative added to foodstuffs. Structural analysis of the 174A bacteriocin using a program that predicts secondary structure suggests that both component polypeptides have a positively charged N-terminal region, as well as two cysteine residues in both the N- and C-terminals. Judging from a mutational analysis of the antibacterial polypeptides, these unique amino acid sequences of 174A-ß might be important for the expression of the synergistic activity that occurs in the presence of the two polypeptides combined.


Subject(s)
Anti-Bacterial Agents/analysis , Bacteriocins/analysis , Citrus/microbiology , Fruit/microbiology , Levilactobacillus brevis/metabolism , Peptides/analysis , Amino Acid Sequence , Anti-Bacterial Agents/pharmacology , Bacteriocins/pharmacology , DNA Mutational Analysis , Drug Synergism , Genes, Bacterial , Lactic Acid , Levilactobacillus brevis/genetics , Molecular Sequence Data , Mutation , Peptides/pharmacology
12.
J Bacteriol ; 195(8): 1741-9, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23396912

ABSTRACT

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.


Subject(s)
Acetyltransferases/metabolism , Bacterial Proteins/metabolism , Cycloserine/biosynthesis , Serine/metabolism , Streptomyces/enzymology , Bacterial Proteins/genetics , Crystallography, X-Ray , DNA-Binding Proteins , Gene Expression Regulation, Bacterial/physiology , Gene Expression Regulation, Enzymologic/physiology , Models, Molecular , Molecular Structure , Mutagenesis, Site-Directed , Protein Conformation , Substrate Specificity , Viral Proteins
13.
Proteins ; 81(11): 2052-8, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23836494

ABSTRACT

Tannin acylhydrolase (EC 3.1.1.20) referred commonly as tannase catalyzes the hydrolysis of the galloyl ester bond of tannins to release gallic acid. Although the enzyme is useful for various industries, the tertiary structure is not yet determined. In this study, we determined the crystal structure of tannase produced by Lactobacillus plantarum. The tannase structure belongs to a member of α/ß-hydrolase superfamily with an additional "lid" domain. A glycerol molecule derived from cryoprotectant solution was accommodated into the tannase active site. The binding manner of glycerol to tannase seems to be similar to that of the galloyl moiety in the substrate.


Subject(s)
Carboxylic Ester Hydrolases/chemistry , Carboxylic Ester Hydrolases/metabolism , Lactobacillus plantarum/enzymology , Carboxylic Ester Hydrolases/genetics , DNA Mutational Analysis , Tannins/metabolism
14.
Antimicrob Agents Chemother ; 57(6): 2603-12, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23529730

ABSTRACT

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.


Subject(s)
Antitubercular Agents/metabolism , Cycloserine/metabolism , Ligases/metabolism , Multigene Family , Serine/metabolism , Streptomyces/enzymology , Biosynthetic Pathways , Chromatography, Thin Layer , Ligases/genetics , Streptomyces/genetics , Substrate Specificity
15.
J Biol Chem ; 286(34): 30219-31, 2011 Aug 26.
Article in English | MEDLINE | ID: mdl-21730070

ABSTRACT

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.


Subject(s)
Bacterial Proteins/chemistry , Copper/chemistry , Metalloproteins/chemistry , Molecular Chaperones/chemistry , Monophenol Monooxygenase/chemistry , Streptococcus/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Biological Transport , Copper/metabolism , Crystallography, X-Ray , Metalloproteins/genetics , Metalloproteins/metabolism , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Monophenol Monooxygenase/genetics , Monophenol Monooxygenase/metabolism , Mutation , Protein Structure, Quaternary , Streptococcus/genetics
16.
Antimicrob Agents Chemother ; 56(7): 3682-9, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22547619

ABSTRACT

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.


Subject(s)
Cycloserine/biosynthesis , Hemeproteins/metabolism , Streptomyces/enzymology , Streptomyces/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Hemeproteins/genetics , Hydroxylation , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/metabolism
17.
Protein Sci ; 31(6): e4338, 2022 06.
Article in English | MEDLINE | ID: mdl-35634777

ABSTRACT

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.


Subject(s)
Arginase , Arginine , Amino Acids , Arginase/chemistry , Arginase/genetics , Arginine/metabolism , Catalysis , Kinetics , Ornithine
18.
J Biol Chem ; 285(2): 1446-56, 2010 Jan 08.
Article in English | MEDLINE | ID: mdl-19889644

ABSTRACT

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.


Subject(s)
Acetyl Coenzyme A/chemistry , Acetyltransferases/chemistry , Bacterial Proteins/chemistry , Bleomycin/chemistry , Streptomyces/enzymology , Catalysis , Crystallography, X-Ray , Protein Structure, Tertiary/physiology
19.
Int J Biol Macromol ; 183: 1861-1870, 2021 Jul 31.
Article in English | MEDLINE | ID: mdl-34089758

ABSTRACT

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.


Subject(s)
Catechol Oxidase/chemistry , Catechol Oxidase/metabolism , Monophenol Monooxygenase/chemistry , Monophenol Monooxygenase/metabolism , Streptomyces/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Catalysis , Catalytic Domain , Catechol Oxidase/genetics , Crystallography, X-Ray , Hydrogen Bonding , Metallochaperones/metabolism , Models, Molecular , Monophenol Monooxygenase/genetics , Mutation , Protein Binding , Protein Conformation , Streptomyces/genetics , Substrate Specificity , Water/chemistry
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