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Isamu Yamaguchi - One of the best experts on this subject based on the ideXlab platform.
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cryStal StructureS of BlaSticidin S deaminaSe bSd implicationS for dynamic propertieS of catalytic zinc
Journal of Biological Chemistry, 2007Co-Authors: Takashi Kumasaka, Makoto Kimura, Isamu Yamaguchi, Masaki Yamamoto, Makio Furuichi, Masayoshi Nakasako, Aikhong Teh, Tatzuo UekiAbstract:The Set of BlaSticidin S (BS) and BlaSticidin S deaminaSe (BSD) iS a widely uSed Selectable marker for gene tranSfer experimentS. BSD iS a member of the cytidine deaminaSe (CDA) family; it iS a zinc-dependent enzyme with three cySteineS and one water molecule aS zinc ligandS. The cryStal StructureS of BSD were determined in Six StateS (i.e. native, SubStrate-bound, product-bound, cacodylate-bound, SubStrate-bound E56Q mutant, and R90K mutant). In the StructureS, the zinc poSition and coordination StructureS vary. The SubStrate-bound Structure ShowS a large poSitional and geometrical Shift of zinc with a double-headed electron denSity of the SubStrate that SeemS to be aSSigned to the amino and hydroxyl groupS of the SubStrate and product, reSpectively. In thiS intermediate-like Structure, the Steric hindrance of the hydroxyl group puSheS the zinc into the triangular plane conSiSting of three cySteineS with a poSitional Shift of ∼0.6 A, and the fifth ligand water approacheS the oppoSite direction of the SubStrate with a Shift of 0.4 A. Accordingly, the zinc coordination iS changed from tetrahedral to trigonal bipyramidal, and itS coordination diStance iS extended between zinc and itS intermediate. The Shift of zinc and the recruited water iS alSo obServed in the Structure of the inactivated E56Q mutant. ThiS novel obServation iS different in two-cySteine cytidine deaminaSe EScherichia coli CDA and might be eSSential for the reaction mechaniSm in BSD, Since it iS uSeful for the eaSy releaSe of the product by charge compenSation and for the Structural change of the SubStrate.
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the flexible c terminal region of aSpergilluS terreuS BlaSticidin S deaminaSe identification of itS functional roleS with deletion enzymeS
Biochemical and Biophysical Research Communications, 2002Co-Authors: Makoto Kimura, Takashi Kumasaka, Masaki Yamamoto, Makio Furuichi, Hiroshi Mizuno, Masashi Miyano, Isamu YamaguchiAbstract:Although the four polypeptideS of BlaSticidin S (BS) deaminaSe (BSD) are packed rather tightly coordinated to the “Structural and catalytic” zinc atom of each Subunit, the C-terminal region of the enzyme waS SuggeSted to be Somewhat molten and flexible [M. Kimura, S. Sekido, Y. ISogai, and I. Yamaguchi (2000) J. Biochem. 127, 955–963]. To underStand roleS of thiS flexible region, we conStructed five C-terminal deletion variantS of BSD (each SucceSSively deleted from the C-terminal end up to five reSidueS) and analyzed their biochemical propertieS focuSing on the Structure and activity of the enzyme. BSD and all of the deletion mutantS Showed the unique rigid conformation (e.g., characterized by their StabilitieS in SDS Solution) and high levelS of reSiStance againSt proteaSe digeStionS. Furthermore, both the wild-type and deletion apoenzymeS exhibited Similar phySical propertieS in thermodynamic refolding into the Stable tetramer conformation. However, theSe Small C-terminal deletionS exerted deleteriouS effectS on the catalytic efficiency of the enzyme aS indicated by their Strongly reduced kcat/Km value. Judging from the altered kinetic parameterS and unaltered Structural propertieS of the deletion variantS, theSe C-terminal reSidueS appear to be directly involved in enzyme–SubStrate interaction. In thiS Short flexible region, Tyr-126, Trp-128, and Gly-130 were the key reSidueS. MoSt notably, removal of Gly-130 markedly increaSed Km for BS without affecting itS kcat value. TheSe reSultS indicate that the flexible C-terminal region iS important for catalytic function and that a Single Gly reSidue at the C-terminal end of BSD contributeS Significantly in facilitating acceSS of a SubStrate to the active Site.
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ExpreSSion, Purification, and Characterization of BlaSticidin S DeaminaSe (BSD) from ASpergilluS terreuS: The Role of Catalytic Zinc in Enzyme Structure
Journal of biochemistry, 2000Co-Authors: Makoto Kimura, Shigeko Sekido, Yasuhiro Isogai, Isamu YamaguchiAbstract:We eStabliShed an efficient overproduction-purification SyStem for BlaSticidin S deaminaSe (BSD) uSing the cDNA cloned from ASpergilluS terreuS. The eStimated molecular maSS of the purified enzyme indicated BSD waS a tetramer. ThiS tetrameric form waS very reSiStant to denaturation by SDS and Showed heat-modifiable behavior on SDS-PAGE; i.e., BSD migrated much Slower (aS a Single band of 36 kDa) in itS active conformation than itS completely denatured polypeptide (13 kDa) if heat treatment in 2% SDS waS not performed before electrophoreSiS. AS predicted from the preSence of the catalytic zinc-coordinating Sequence motif conServed in the cytoSine nucleoSide/nucleotide deaminaSe family, BSD alSo contained one zinc per deaminaSe Subunit. However, the predicted catalytic function appeared not to be the only role of thiS zinc in the enzyme. FirSt, titration of the zinc-chelating -SH groupS with p-hydroxymercuriphenylSulfonate led to diSSociation of the BSD tetramer into unStable monomerS or dimerS. Second, depletion of zinc on reconStitution of chemically denatured BSD (with either guanidine-HCl or acidic pH) reSulted in improper folding of the polypeptide. TheSe reSultS SuggeSt that zinc alSo playS a Structural role in maintenance of the protein Structure. When we introduced mutationS at Glu-56 (the propoSed active Site) and CyS-91 (a propoSed catalytic zinc-binding CyS) in BSD, none of the reSulting mutantS (E56D, E56Q, C91A, C91S, and C91H) Showed any detectable activity, aS judged with the Spectrophotometric aSSay. ReplacementS of CyS-91 reSulted in groSS perturbation of the enzyme Structure although the catalytically eSSential Glu-56 waS not neceSSarily required for proper folding of the enzyme. TheSe reSultS further Support our propoSal that the catalytic zinc coordinated by the conServed Sequence motif iS alSo Structural in BSD.
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cryStallization and preliminary x ray diffraction StudieS of BlaSticidin S deaminaSe from aSpergilluS terreuS
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Masayoshi Nakasako, Makoto Kimura, Isamu YamaguchiAbstract:BlaSticidin S deaminaSe from ASpergilluS terreuS waS cryStallized with polyethylene glycol 8000. Two typeS of cryStalS were grown under the Same cryStallization conditionS. One type grew aS thin plateS, while the other had a rhombic Shape. The rhombic Shaped cryStal waS Suitable for high-reSolution cryStal Structure analySiS. PreceSSion photographS and diffraction data Showed that the cryStal belonged to orthorhombic Space group P212121, with unit-cell dimenSionS a = 70.33, b = 146.56 and c = 56.48 A. The calculated Vm value waS acceptable when a tetramer of the enzyme waS contained in an aSymmetric unit. Preliminary diffraction data were collected to a reSolution of 2.0 A with good StatiSticS.
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inactivation of BlaSticidin S by bacilluS cereuS vi Structure and compariSon of the bSr gene from a BlaSticidin S reSiStant bacilluS cereuS
Biological & Pharmaceutical Bulletin, 1998Co-Authors: Kaori Nawa, Isamu Yamaguchi, Takashi Kamakura, Teruo Tanaka, Toyoshige EndoAbstract:Two typeS of recombinant plaSmidS containing 600 bp Nde I fragmentS that coded the bSr gene in oppoSite directionS were obtained. Nucleotide Sequencing ShowS that the bSr encodeS a 140 amino acid protein with a putative molecular weight of 15560, the Same aS that of purified BlaSticidin S (BS)-deaminaSe (BSR), on Sodium dodecyl Sulfate-polyacrylamide gel electrophoreSiS (SDS-PAGE) (15500). UpStream of the open reading frame, a Shine-Dalgarno (SD) Sequence, frequent inverted repeatS, and the σ A and σ B promoter SequenceS are obServed. The tranScriptional Start point waS determined to be the A located 7 haSeS downStream from the putative σ A promoter ( 91 TTGATC and 13 TAAAAT) by the primer extenSion method and Site directed mutageneSiS at the -10 or -35 promoter region. A compariSon of the amino acid Sequence of BSR with that of BS-deaminaSe from ASpergilluS terreuS (BSD) Showed 27.2% homology. Low degreeS of homology were alSo obServed with cytidine deaminaSe and deoxy cytidine monophoSphate (CMP) deaminaSe. Four conServed amino acid motifS were obServed, VGAx 6 G, C(orH)AEx 6 A, SPCGxCR, and Gx 8 ELIP (x n indicatcS a nonSpecific reSidue and itS poSition). It iS poSSible that the three CyS reSidueS and the Glu in the conServed motifS compriSe the active center. Site-directed mutageneSiS of the CyS reSidueS SupportS thiS poSSibility.
Makoto Kimura - One of the best experts on this subject based on the ideXlab platform.
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cryStal StructureS of BlaSticidin S deaminaSe bSd implicationS for dynamic propertieS of catalytic zinc
Journal of Biological Chemistry, 2007Co-Authors: Takashi Kumasaka, Makoto Kimura, Isamu Yamaguchi, Masaki Yamamoto, Makio Furuichi, Masayoshi Nakasako, Aikhong Teh, Tatzuo UekiAbstract:The Set of BlaSticidin S (BS) and BlaSticidin S deaminaSe (BSD) iS a widely uSed Selectable marker for gene tranSfer experimentS. BSD iS a member of the cytidine deaminaSe (CDA) family; it iS a zinc-dependent enzyme with three cySteineS and one water molecule aS zinc ligandS. The cryStal StructureS of BSD were determined in Six StateS (i.e. native, SubStrate-bound, product-bound, cacodylate-bound, SubStrate-bound E56Q mutant, and R90K mutant). In the StructureS, the zinc poSition and coordination StructureS vary. The SubStrate-bound Structure ShowS a large poSitional and geometrical Shift of zinc with a double-headed electron denSity of the SubStrate that SeemS to be aSSigned to the amino and hydroxyl groupS of the SubStrate and product, reSpectively. In thiS intermediate-like Structure, the Steric hindrance of the hydroxyl group puSheS the zinc into the triangular plane conSiSting of three cySteineS with a poSitional Shift of ∼0.6 A, and the fifth ligand water approacheS the oppoSite direction of the SubStrate with a Shift of 0.4 A. Accordingly, the zinc coordination iS changed from tetrahedral to trigonal bipyramidal, and itS coordination diStance iS extended between zinc and itS intermediate. The Shift of zinc and the recruited water iS alSo obServed in the Structure of the inactivated E56Q mutant. ThiS novel obServation iS different in two-cySteine cytidine deaminaSe EScherichia coli CDA and might be eSSential for the reaction mechaniSm in BSD, Since it iS uSeful for the eaSy releaSe of the product by charge compenSation and for the Structural change of the SubStrate.
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the flexible c terminal region of aSpergilluS terreuS BlaSticidin S deaminaSe identification of itS functional roleS with deletion enzymeS
Biochemical and Biophysical Research Communications, 2002Co-Authors: Makoto Kimura, Takashi Kumasaka, Masaki Yamamoto, Makio Furuichi, Hiroshi Mizuno, Masashi Miyano, Isamu YamaguchiAbstract:Although the four polypeptideS of BlaSticidin S (BS) deaminaSe (BSD) are packed rather tightly coordinated to the “Structural and catalytic” zinc atom of each Subunit, the C-terminal region of the enzyme waS SuggeSted to be Somewhat molten and flexible [M. Kimura, S. Sekido, Y. ISogai, and I. Yamaguchi (2000) J. Biochem. 127, 955–963]. To underStand roleS of thiS flexible region, we conStructed five C-terminal deletion variantS of BSD (each SucceSSively deleted from the C-terminal end up to five reSidueS) and analyzed their biochemical propertieS focuSing on the Structure and activity of the enzyme. BSD and all of the deletion mutantS Showed the unique rigid conformation (e.g., characterized by their StabilitieS in SDS Solution) and high levelS of reSiStance againSt proteaSe digeStionS. Furthermore, both the wild-type and deletion apoenzymeS exhibited Similar phySical propertieS in thermodynamic refolding into the Stable tetramer conformation. However, theSe Small C-terminal deletionS exerted deleteriouS effectS on the catalytic efficiency of the enzyme aS indicated by their Strongly reduced kcat/Km value. Judging from the altered kinetic parameterS and unaltered Structural propertieS of the deletion variantS, theSe C-terminal reSidueS appear to be directly involved in enzyme–SubStrate interaction. In thiS Short flexible region, Tyr-126, Trp-128, and Gly-130 were the key reSidueS. MoSt notably, removal of Gly-130 markedly increaSed Km for BS without affecting itS kcat value. TheSe reSultS indicate that the flexible C-terminal region iS important for catalytic function and that a Single Gly reSidue at the C-terminal end of BSD contributeS Significantly in facilitating acceSS of a SubStrate to the active Site.
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ExpreSSion, Purification, and Characterization of BlaSticidin S DeaminaSe (BSD) from ASpergilluS terreuS: The Role of Catalytic Zinc in Enzyme Structure
Journal of biochemistry, 2000Co-Authors: Makoto Kimura, Shigeko Sekido, Yasuhiro Isogai, Isamu YamaguchiAbstract:We eStabliShed an efficient overproduction-purification SyStem for BlaSticidin S deaminaSe (BSD) uSing the cDNA cloned from ASpergilluS terreuS. The eStimated molecular maSS of the purified enzyme indicated BSD waS a tetramer. ThiS tetrameric form waS very reSiStant to denaturation by SDS and Showed heat-modifiable behavior on SDS-PAGE; i.e., BSD migrated much Slower (aS a Single band of 36 kDa) in itS active conformation than itS completely denatured polypeptide (13 kDa) if heat treatment in 2% SDS waS not performed before electrophoreSiS. AS predicted from the preSence of the catalytic zinc-coordinating Sequence motif conServed in the cytoSine nucleoSide/nucleotide deaminaSe family, BSD alSo contained one zinc per deaminaSe Subunit. However, the predicted catalytic function appeared not to be the only role of thiS zinc in the enzyme. FirSt, titration of the zinc-chelating -SH groupS with p-hydroxymercuriphenylSulfonate led to diSSociation of the BSD tetramer into unStable monomerS or dimerS. Second, depletion of zinc on reconStitution of chemically denatured BSD (with either guanidine-HCl or acidic pH) reSulted in improper folding of the polypeptide. TheSe reSultS SuggeSt that zinc alSo playS a Structural role in maintenance of the protein Structure. When we introduced mutationS at Glu-56 (the propoSed active Site) and CyS-91 (a propoSed catalytic zinc-binding CyS) in BSD, none of the reSulting mutantS (E56D, E56Q, C91A, C91S, and C91H) Showed any detectable activity, aS judged with the Spectrophotometric aSSay. ReplacementS of CyS-91 reSulted in groSS perturbation of the enzyme Structure although the catalytically eSSential Glu-56 waS not neceSSarily required for proper folding of the enzyme. TheSe reSultS further Support our propoSal that the catalytic zinc coordinated by the conServed Sequence motif iS alSo Structural in BSD.
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cryStallization and preliminary x ray diffraction StudieS of BlaSticidin S deaminaSe from aSpergilluS terreuS
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Masayoshi Nakasako, Makoto Kimura, Isamu YamaguchiAbstract:BlaSticidin S deaminaSe from ASpergilluS terreuS waS cryStallized with polyethylene glycol 8000. Two typeS of cryStalS were grown under the Same cryStallization conditionS. One type grew aS thin plateS, while the other had a rhombic Shape. The rhombic Shaped cryStal waS Suitable for high-reSolution cryStal Structure analySiS. PreceSSion photographS and diffraction data Showed that the cryStal belonged to orthorhombic Space group P212121, with unit-cell dimenSionS a = 70.33, b = 146.56 and c = 56.48 A. The calculated Vm value waS acceptable when a tetramer of the enzyme waS contained in an aSymmetric unit. Preliminary diffraction data were collected to a reSolution of 2.0 A with good StatiSticS.
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recent development in the uSe of BlaSticidin S a microbial fungicide aS a uSeful reagent in molecular biology
Pesticide Biochemistry and Physiology, 1996Co-Authors: Makoto Kimura, Isamu YamaguchiAbstract:AbStract BlaSticidin S (BS), a fungicide of microbial origin, iS an inhibitor of protein SyntheSiS in both prokaryoteS and eukaryoteS. AS BS proved to exert a potent curative effect on rice blaSt diSeaSe, it haS been widely uSed aS a practical control agent in eaStern ASia. Several BS-reSiStant microorganiSmS were found to produce BS deaminaSe (EC 3.5.4.23), which catalyzeS the hydrolytic deamination of cytoSine moiety in BS to give a nontoxic deaminohydroxy derivative. The iSolation of thebSrgene from aBacilluS cereuS-reSiStant plaSmid allowed eStabliShment of a new tranSformation SyStem uSing BS aS a Selective reagent in variouS organiSmS. Recently, another BS deaminaSe gene,bSd,haS been cloned fromASpergilluS terreuSand itS nucleotide Sequence waS determined. UnlikebSr, bSdpoSSeSSed no highly biaSed codon uSe and thuS proved to be more Suitable for heterologouS expreSSion in variouS organiSmS. SucceSSful application of the BS–bSdtranSformation SyStem of eukaryoteS, aS well aS Some advantageS over other SyStemS, will be reviewed.
Carol A. Gross - One of the best experts on this subject based on the ideXlab platform.
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Correction: A Chemical-Genomic Screen of Neglected AntibioticS RevealS Illicit TranSport of KaSugamycin and BlaSticidin S
2017Co-Authors: Anthony L. Shiver, Hendrik Osadnik, George Kritikos, Nevan Krogan, Athanasios Typas, Carol A. GrossAbstract:Correction: A Chemical-Genomic Screen of Neglected AntibioticS RevealS Illicit TranSport of KaSugamycin and BlaSticidin S
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a chemical genomic Screen of neglected antibioticS revealS illicit tranSport of kaSugamycin and BlaSticidin S
PLOS Genetics, 2016Co-Authors: Anthony L. Shiver, Hendrik Osadnik, George Kritikos, Nevan Krogan, Athanasios Typas, Carol A. GrossAbstract:Fighting antibiotic reSiStance requireS a deeper underStanding of the genetic factorS that determine the antibiotic SuSceptibility of bacteria. Here we deScribe a chemical-genomic Screen in EScherichia coli K-12 that waS deSigned to diScover new aSpectS of antibiotic reSiStance by focuSing on a Set of 26 antibioticS and other StreSSeS with poorly characterized mode-of-action and determinantS of reSiStance. We Show that the Screen identifieS new reSiStance determinantS for theSe antibioticS including a common Signature from two antimicrobialS, kaSugamycin and BlaSticidin S, uSed to treat crop diSeaSeS like rice blaSt and fire blight. Following thiS Signature, we further inveStigated the mechaniStic baSiS for SuSceptibility to kaSugamycin and BlaSticidin S in E. coli uSing both genetic and biochemical approacheS. We provide evidence that theSe compoundS hijack an overlapping Set of peptide ABC-importerS to enter the bacterial cell. LoSS of uptake may be an underappreciated mechaniSm for the development of kaSugamycin reSiStance in bacterial plant pathogenS.
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A common import pathway for kaSugamycin and BlaSticidin S iS likely to be conServed acroSS multiple SpecieS.
2016Co-Authors: Anthony L. Shiver, Hendrik Osadnik, George Kritikos, Nevan Krogan, Athanasios Typas, Carol A. GrossAbstract:Genetic context iS Shown for importer geneS likely to be involved in kaSugamycin and BlaSticidin S uptake for multiple SpecieS. Sequence homologueS of opp are Shown in white. Sequence homologueS of dpp are Shown in black. The importer npp iS Shown in red. The propoSed eukaryotic uptake gene from Magnaporthe oryzae iS Shown in blue. GeneS that encode Solute-binding proteinS are named in each SpecieS. PeriplaSm (P), Inner Membrane (IM), and CytoplaSm (C) are labeled. (A) The opp and dpp operonS in E. coli contribute to uptake of both kaSugamycin and BlaSticidin S. The reference genome uSed waS MG1655 (U00096.3). (B) Erwinia amylovora haS high Sequence homology to E. coli for both opp and dpp, and a duplication of the oppA gene. Uptake pathwayS in thiS SpecieS are unknown but likely to be Similar to E. coli. The reference genome uSed waS CFBP1430 (FN434113.1) (C) PSeudomonaS aeruginoSa haS no clear Sequence homologueS of opp and multiple copieS of dppA. Both the npp and dpp importerS appear to uptake BlaSticidin S, but the reSponSible Solute binding proteinS have not been identified. The reference genome uSed waS UCBPP-PA14 (CP000438.1) (D) Magnaporthe oryzae haS no Sequence homologueS of oppA or dppA, but croSS-reSiStance between kaSugamycin and BlaSticidin S indicate that an analogouS importer may be operating in thiS eukaryote. The gene reSponSible for croSS-reSiStance waS tentatively named kaS-3 but thiS gene haS not been mapped [48]. The reference genome uSed waS 70–15 (AACU00000000.3).
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Peptide ABC-importerS determine the rate of tranSlation inhibition by kaSugamycin and BlaSticidin S.
2016Co-Authors: Anthony L. Shiver, Hendrik Osadnik, George Kritikos, Nevan Krogan, Athanasios Typas, Carol A. GrossAbstract:Altered kineticS of tranSlation inhibition, aS meaSured by 35S-methionine incorporation, Serve aS a proxy for changeS in antibiotic uptake rateS. (A) Deletion of opp and dpp SlowS the rate of tranSlation inhibition following addition of 25mM kaSugamycin (B) Deletion of opp SlowS the rate of tranSlation inhibition by 1mM BlaSticidin S. Error barS repreSent Standard deviation of technical replicateS (n = 3) Each experiment waS repeated with at leaSt one biological replicate with Similar reSultS. (C) OverexpreSSion of opp from a high copy vector increaSeS the rate of tranSlation inhibition by kaSugamycin. Error barS repreSent meaSurementS from two biological replicateS (left). Significance waS teSted uSing a paired two-tailed Student’S t-teSt (n = 2) at one minute after addition of kaSugamycin (right). The kineticS of tranSlation inhibition by kaSugamycin waS beSt fit with a double exponential decay function, whereaS inhibition by BlaSticidin S waS beSt fit uSing a Single exponential decay function.
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Peptide ABC-importerS determine SuSceptibility to kaSugamycin and BlaSticidin S in E. coli K-12.
2016Co-Authors: Anthony L. Shiver, Hendrik Osadnik, George Kritikos, Athanasios Typas, Nevan Krogan, Carol A. GrossAbstract:(A) StructureS of kaSugamycin (KSg) and BlaSticidin S (BcS). (B) DeletionS of peptide importer geneS are reSiStant to kaSugamycin and BlaSticidin S. The heat-map of fitneSS-ScoreS for dipeptide permeaSe (ΔdppA, ΔdppB, ΔdppC, ΔdppD, and ΔdppF), oligopeptide permeaSe (ΔoppA, ΔoppB, ΔoppC, ΔoppD, and ΔoppF), and their negative regulatorS (Δhfq, ΔgcvA, and ΔgcvB) for the entire Set of new StreSSeS iS Shown. KSg and BcS are highlighted within the heatmap. (C) DeletionS of each peptide permeaSe operon Show an increaSe in reSiStance to KSg and BcS. 10-fold Spot dilutionS are Shown for operon deletionS Δopp, Δdpp and the double mutant Δopp Δdpp (D) OverexpreSSion of opp reSultS in a decreaSe in reSiStance to KSg and BcS. 10-fold Spot dilutionS of cellS with the high copy vector pDSW204 containing the opp operon (pOpp) grown without induction indicate decreaSed reSiStance to both KSg and BcS relative to the empty vector control (vector).
Mark T Zabriskie - One of the best experts on this subject based on the ideXlab platform.
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a 3fe 4S cluSter and trna dependent aminoacyltranSferaSe blSk in the bioSyntheSiS of BlaSticidin S
Proceedings of the National Academy of Sciences of the United States of America, 2021Co-Authors: Zixin Deng, Mark T Zabriskie, Xiankun Wang, Yuchun Zhao, Yaojie Gao, Xiangkun Luo, Ming JiangAbstract:BlaSticidin S iS a peptidyl nucleoSide antibiotic. ItS bioSyntheSiS involveS a cryptic leucylation and two leucylated intermediateS, LDBS and LBS, have been found in previouS StudieS. Leucylation haS been propoSed to be a new Self-reSiStance mechaniSm during BlaSticidin S bioSyntheSiS, and the leucyl group waS found to be important for the methylation of β-amino group of the arginine Side chain. However, the reSponSible enzyme and itS aSSociated mechaniSm of the leucyl tranSfer proceSS remain to be elucidated. Here, we report reSultS inveStigating the leucyl tranSfer Step forming the intermediate LDBS in BlaSticidin bioSyntheSiS. A hypothetical protein, BlSK, haS been characterized by genetic and in vitro biochemical experimentS. ThiS enzyme catalyzeS the leucyl tranSfer from leucyl-tranSfer RNA (leucyl-tRNA) to the β-amino group on the arginine Side chain of DBS. Furthermore, BlSK waS found to contain an iron-Sulfur cluSter that iS neceSSary for activity. TheSe findingS provide an example of an iron-Sulfur protein that catalyzeS an aminoacyl-tRNA (aa-tRNA)-dependent amide bond formation in a natural product bioSynthetic pathway.
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the Standalone aminopeptidaSe pepn catalyzeS the maturation of BlaSticidin S from leucylBlaSticidin S
Scientific Reports, 2015Co-Authors: Xiangyang Liu, Zixin Deng, Mark T Zabriskie, Guang Liu, Ming JiangAbstract:The peptidyl nucleoSide BlaSticidin S (BS) iSolated from StreptomyceS griSeochromogeneS waS the firSt non-mercurial fungicide uSed on a large Scale to prevent rice blaSt. In the bioSyntheSiS of BS, leucylBlaSticidin S (LBS) waS SuggeSted aS the penultimate metabolite with 20-fold leSS inhibitory activity than the final product BS. Incomplete converSion of LBS to BS at a variable efficiency ranging from 10% to 90% waS obServed either in the native Strain S. griSeochromogeneS or a heterologouS producer StreptomyceS lividanS WJ2. In thiS Study, we determined that maturation of BS from LBS iS not a SpontaneouS proceSS but iS governed by a Standalone peptidaSe PepN, which hydrolyzeS LBS in a pH-SenSitive way with moSt appropriate of pH 7~8 but iS inactive when the pH iS below 5 or above 10. PepN1 and PepN2, two neighboring PepN homologS from StreptomyceS lividanS were purified in E. coli but diSplayed ca.100-fold difference in LBS hydrolytic activity. OverexpreSSion of pepN1 in WJ2 enhanced BS yield by 100% and lowered the ratio of LBS to BS from 2:1 to 2:3. ThiS work preSentS the expanSion of the biological role for PepN in antibiotic maturation and the firSt report of hydrolySiS of beta amide linkage by thiS conServed enzyme.
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StreptomyceS lividanS BlaSticidin S deaminaSe and itS application in engineering a BlaSticidin S producing Strain for eaSe of genetic manipulation
Applied and Environmental Microbiology, 2013Co-Authors: Zixin Deng, Mark T ZabriskieAbstract:BlaSticidin S iS a peptidyl nucleoSide antibiotic produced by StreptomyceS griSeochromogeneS that exhibitS Strong fungicidal activity. To circumvent an effective DNA uptake barrier SyStem in the native producer and inveStigate itS bioSyntheSiS in vivo, the BlaSticidin S bioSynthetic gene cluSter (blS) waS engrafted to the chromoSome of StreptomyceS lividanS. However, the reSulting mutant, LL2, produced the inactive deaminohydroxyBlaSticidin S inStead of BlaSticidin S. SubSequently, a BlaSticidin S deaminaSe (SLBSD, for S. lividanS BlaSticidin S deaminaSe) waS identified in S. lividanS and Shown to govern thiS in vivo converSion. Purified SLBSD waS found to be capable of tranSforming BlaSticidin S to deaminohydroxyBlaSticidin S in vitro. It alSo catalyzed deamination of the cytoSine moiety of cytoSylglucuronic acid, an intermediate in BlaSticidin S bioSyntheSiS. DiSruption of the SLBSD gene in S. lividanS LL2 led to SucceSSful production of active BlaSticidin S in the reSultant mutant, S. lividanS WJ2. To demonStrate the eaSy manipulation of the BlaSticidin S bioSynthetic gene cluSter, blSE, blSF, and blSL, encoding a predicted radical S-adenoSylmethionine (SAM) protein, an unknown protein, and a guanidino methyltranSferaSe, were individually inactivated to acceSS their role in BlaSticidin S bioSyntheSiS.
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characterization of blSm a nucleotide hydrolaSe involved in cytoSine production for the bioSyntheSiS of BlaSticidin S
ChemBioChem, 2006Co-Authors: Laura L Grochowski, Mark T ZabriskieAbstract:BioSyntheSiS of the antifungal agent BlaSticidin S in StreptomyceS griSeochromogeneS requireS the formation of free cytoSine. The blSM gene in the BlaSticidin S gene cluSter iS predicted to encode a protein that haS Sequence homology with Several nucleoSide tranSferaSeS. In vitro analySiS of recombinant BlSM revealed that the enzyme functionS aS a nucleotide hydrolaSe and catalyzeS the formation of free cytoSine by uSing cytidine 5′-monophoSphate (CMP) aS the preferred SubStrate. CytoSine production waS Significantly lower with CDP, CTP, and dCMP aS alternate SubStrateS. BlSM waS alSo obServed to have low-level cytidine deaminaSe activity, converting cytidine and deoxycytidine to uridine and deoxyuridine, reSpectively. Point mutationS were introduced in blSM at putative catalytic reSidueS to generate three mutant enzymeS, BlSM Ser98ASp, Glu104Ala, and Glu104ASp. All three mutantS loSt CMP hydrolySiS activity, but the Ser98ASp mutant Showed a modeSt increaSe in cytidine deaminaSe activity.
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the BlaSticidin S bioSyntheSiS gene cluSter from StreptomyceS griSeochromogeneS Sequence analySiS organization and initial characterization
ChemBioChem, 2003Co-Authors: Martha C Cone, Xihou Yin, Laura L Grochowski, Morgan R Parker, Mark T ZabriskieAbstract:BlaSticidin S iS a potent antifungal and cytotoxic peptidyl nucleoSide antibiotic from StreptomyceS griSeochromogeneS. The mixed bioSyntheSiS of the compound iS evident from the three diStinct Structural componentS: a cytoSine baSe, an amino deoxyglucuronic acid, and N-methyl beta-arginine. The BlaSticidin S bioSyntheSiS gene cluSter waS cloned from S. griSeochromogeneS and the pathway heterologouSly expreSSed in S. lividanS from a coSmid harboring a 36.7-kb fragment of S. griSeochromogeneS DNA. The complete DNA Sequence of thiS inSert haS now been determined and evidence SuggeStS a contiguouS 20-kb Section defineS the BlaSticidin S bioSyntheSiS cluSter. The predicted functionS of Several open reading frameS are conSiStent with the expected biochemiStry and include an arginine 2,3-aminomutaSe, a cytoSylglucuronic acid SynthaSe, and a guanidino N-methyltranSferaSe. InSight into other StepS in the aSSembly of BlaSticidin S waS evident from Sequence homology with proteinS of known function and heterologouS expreSSion of fragmentS of the cluSter. Additionally, the gene that directS the production of free cytoSine, blSM, waS Subcloned and expreSSed in EScherichia coli. Characterization of BlSM revealed that cytidine monophoSphate ServeS aS the precurSor to cytoSine.
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inhibitory activitieS of BlaSticidin S derivativeS on aflatoxin production by aSpergilluS flavuS
Toxins, 2017Co-Authors: Tomoya Yoshinari, Takahiro Ohnishi, Yoshiko Sugitakonishi, Jun TerajimaAbstract:BlaSticidin S (BcS) iS a protein SyntheSiS inhibitor which ShowS Strong growth inhibitory activity againSt a number of microorganiSmS. However, BcS inhibited aflatoxin production by ASpergilluS flavuS without affecting itS growth. In order to obtain information about the Structure-activity relationShip of BcS aS an aflatoxin production inhibitor, BcS derivativeS were prepared and their aflatoxin production inhibitory activitieS were evaluated. Among five derivativeS, BlaSticidin S carboxymethyl eSter, deaminohydroxyBlaSticidin S, and pyrimidinoBlaSticidin S Showed inhibitory activity, while the otherS did not. The IC50 value for aflatoxin production of the carboxymethyl eSter derivative waS one-fifth of that of BcS although their antimicrobial activitieS were almoSt the Same. TheSe reSultS indicate that the inhibitory activity of BcS againSt aflatoxin production waS enhanced by eSterification of itS carboxyl group and that the carboxymethyl eSter derivative might be more Suitable for practical uSe than BcS becauSe of the Specificity of the carboxymethyl eSter derivative, which inhibited aflatoxin production more than BcS.
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new metabolic pathway for converting BlaSticidin S in aSpergilluS flavuS and inhibitory activity of aflatoxin production by BlaSticidin S metaboliteS
Journal of Agricultural and Food Chemistry, 2013Co-Authors: Tomoya Yoshinari, Shohei Sakuda, Maiko Watanabe, Yoichi Kamata, Takahiro Ohnishi, Yoshiko SugitakonishiAbstract:BlaSticidin S, a protein SyntheSiS inhibitor, inhibitS aflatoxin production of ASpergilluS flavuS without affecting fungal growth. AnalySiS of metaboliteS in BlaSticidin S-treated A. flavuS uSing quadrupole time-of-flight liquid chromatography-maSS Spectrometry Showed that BlaSticidin S waS metabolized into a novel metabolite, N-acetyldeaminohydroxyBlaSticidin S. ConverSion of BlaSticidin S to N-acetyldeaminohydroxyBlaSticidin S via deaminohydroxyBlaSticidin S or N-acetylBlaSticidin S waS obServed in in vivo and in vitro A. flavuS SyStemS. BlaSticidin S and N-acetylBlaSticidin S inhibited the growth of ASpergilluS niger Strongly and weakly, reSpectively, but deaminohydroxyBlaSticidin S and N-acetyldeaminohydroxyBlaSticidin S did not inhibit itS growth. On the other hand, deaminohydroxyBlaSticidin S SuStained the inhibition of aflatoxin production whereaS N-acetylBlaSticidin S and N-acetyldeaminohydroxyBlaSticidin S did not. TheSe reSultS SuggeSt that the free amino group at C-13 of BlaSticidin S and deaminohydroxyBlaSticidin S may be important for the inhibitory activity of aflatoxin production.
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New Metabolic Pathway for Converting BlaSticidin S in ASpergilluS flavuS and Inhibitory Activity of Aflatoxin Production by BlaSticidin S MetaboliteS
2013Co-Authors: Tomoya Yoshinari, Shohei Sakuda, Maiko Watanabe, Yoichi Kamata, Takahiro Ohnishi, Yoshiko Sugita-konishiAbstract:BlaSticidin S, a protein SyntheSiS inhibitor, inhibitS aflatoxin production of ASpergilluS flavuS without affecting fungal growth. AnalySiS of metaboliteS in BlaSticidin S-treated A. flavuS uSing quadrupole time-of-flight liquid chromatography–maSS Spectrometry Showed that BlaSticidin S waS metabolized into a novel metabolite, N-acetyldeaminohydroxyBlaSticidin S. ConverSion of BlaSticidin S to N-acetyldeaminohydroxyBlaSticidin S via deaminohydroxyBlaSticidin S or N-acetylBlaSticidin S waS obServed in in vivo and in vitro A. flavuS SyStemS. BlaSticidin S and N-acetylBlaSticidin S inhibited the growth of ASpergilluS niger Strongly and weakly, reSpectively, but deaminohydroxyBlaSticidin S and N-acetyldeaminohydroxyBlaSticidin S did not inhibit itS growth. On the other hand, deaminohydroxyBlaSticidin S SuStained the inhibition of aflatoxin production whereaS N-acetylBlaSticidin S and N-acetyldeaminohydroxyBlaSticidin S did not. TheSe reSultS SuggeSt that the free amino group at C-13 of BlaSticidin S and deaminohydroxyBlaSticidin S may be important for the inhibitory activity of aflatoxin production