| 1. |
Campos-Olivas R,
Hörr I,
Bormann C,
Jung G,
Gronenborn AM,
( 2001 ) Solution structure, backbone dynamics and chitin binding of the anti-fungal protein from Streptomyces tendae TU901. PMID : 11350173 : DOI : 10.1006/jmbi.2001.4622 Abstract >>
AFP1 is a recently discovered anti-fungal, chitin-binding protein from Streptomyces tendae T?901. Mature AFP1 comprises 86 residues and exhibits limited sequence similarity to the cellulose-binding domains of bacterial cellulases and xylanases. No similarity to the Cys and Gly-rich domains of plant chitin-binding proteins (e.g. agglutinins, lectins, hevein) is observed. AFP1 is the first chitin-binding protein from a bacterium for which anti-fungal activity was shown. Here, we report the three-dimensional solution structure of AFP1, determined by nuclear magnetic resonance spectroscopy. The protein contains two antiparallel beta-sheets (five and four beta-strands each), that pack against each other in a parallel beta-sandwich. This type of architecture is conserved in the functionally related family II of cellulose-binding domains, albeit with different connectivity. A similar fold is also observed in other unrelated proteins (spore coat protein from Myxococcus xanthus, beta-B2 and gamma-B crystallins from Bos taurus, canavalin from Jack bean). AFP1 is therefore classified as a new member of the betagamma-crystallin superfamily. The dynamics of the protein was characterized by NMR using amide 15N relaxation and solvent exchange data. We demonstrate that the protein exhibits an axially symmetric (oblate-like) rotational diffusion tensor whose principal axis coincides to within 15 degrees with that of the inertial tensor. After completion of the present structure of AFP1, an identical fold was reported for a Streptomyces killer toxin-like protein. Based on sequence comparisons and clustering of conserved residues on the protein surface for different cellulose and chitin-binding proteins, we postulate a putative sugar-binding site for AFP1. The inability of the protein to bind short chitin fragments suggests that certain particular architectural features of the solid chitin surface are crucial for the interaction.
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2. |
Süssmuth R,
Kaiser D,
Lauer B,
( 2000 ) A putative enolpyruvyl transferase gene involved in nikkomycin biosynthesis. PMID : 10866220 : DOI : 10.7164/antibiotics.53.385 Abstract >>
The nikO gene encoding a putative enolpyruvyl transferase has been identified within the Streptomyces tendae T?901/8c nikkomycin gene cluster. nikO encodes a deduced protein of 471 amino acid residues which exhibits significant sequence similarity to UDP-N-acetylglucosamine enolpyruvyl transferase and 5-enol-pyruvylshikimate 3-phosphate synthase from various origin. The nikO gene was inactivated by inserting a kanamycin resistance cassette; the mutant did not produce biologically active nikkomycins I, J, X, and Z nor the nucleoside moieties, nikkomycins C(x) and C(z), but accumulated the novel component RT 2.0. RT 2.0 has been isolated from culture filtrate and its structure was determined by using mass spectrometry and NMR analyses as ribofuranosyl-4-formyl-4-imidazolone which represents a novel nucleoside. The putative activity of the nikO gene product in nikkomycin biosynthesis will be discussed.
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3. |
Russwurm R,
Lauer B,
( 2000 ) Molecular characterization of two genes from Streptomyces tendae T?901 required for the formation of the 4-formyl-4-imidazolin-2-one-containing nucleoside moiety of the peptidyl nucleoside antibiotic nikkomycin. PMID : 10712601 : DOI : 10.1046/j.1432-1327.2000.01162.x Abstract >>
The genes nikQ and nikR were identified by sequencing DNA of the nikkomycin biosynthetic gene cluster from Streptomyces tendae T?901/8c. The nikQ gene encodes a P450 cytochrome, and the predicted NikR gene product shows 48-56% sequence identity with uracil phosphoribosyltransferases from eukaryotic organisms. The nikQ and nikR genes were inactivated separately by insertion of a kanamycin-resistance cassette. Inactivation of the nikQ gene abolished synthesis of nikkomycins containing 4-formyl-4-imidazolin-2-one as the base (nikkomycins X and I), whereas production of nikkomycins containing uracil (nikkomycins Z and J) was not affected. Nikkomycin X and I production could be restored by feeding 4-formyl-4-imidazolin-2-one to the nikQ mutants, indicating that NikQ is responsible for its formation from L-histidine. Disruption of the nikR gene resulted in formation of decreased amounts of nikkomycins X and I, whereas nikkomycins Z and J were synthesized at wild-type levels. A fluorouracil-resistant nikR mutant lacking uracil phosphoribosyltransferase (UPRTase) activity did not synthesize nikkomycins X and I and accumulated 4-formyl-4-imidazolin-2-one in its culture filtrate, whereas formation of nikkomycins Z and J was unimpaired. The mutant was complemented to nikkomycin X and I production by nikR expressed from the mel promoter of plasmid pIJ702. The nikR gene expressed in Escherichia coli led to the production of UPRTase activity. Our results indicate that NikR converts 4-formyl-4-imidazolin-2-one to yield 5'-phosphoribosyl-4-formyl-4-imidazolin-2-one, the precursor of nikkomycins containing this base.
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4. |
Hörr I,
Raps C,
Berger J,
Baier D,
Bormann C,
( 1999 ) Characterization of a novel, antifungal, chitin-binding protein from Streptomyces tendae T?901 that interferes with growth polarity. PMID : 10601197 : PMC : PMC94197 Abstract >>
The afp1 gene, which encodes the antifungal protein AFP1, was cloned from nikkomycin-producing Streptomyces tendae T?901, using a nikkomycin-negative mutant as a host and screening transformants for antifungal activity against Paecilomyces variotii in agar diffusion assays. The 384-bp afp1 gene has a low G+C content (63%) and a transcription termination structure with a poly(T) region, unusual attributes for Streptomyces genes. AFP1 was purified from culture filtrate of S. tendae carrying the afp1 gene on the multicopy plasmid pIJ699. The purified protein had a molecular mass of 9,862 Da and lacked a 42-residue N-terminal peptide deduced from the nucleotide sequence. AFP1 was stable at extreme pH values and high temperatures and toward commercial proteinases. AFP1 had limited similarity to cellulose-binding domains of microbial plant cell wall hydrolases and bound to crab shell chitin, chitosan, and cell walls of P. variotii but showed no enzyme activity. The biological activity of AFP1, which represents the first chitin-binding protein from bacteria exhibiting antifungal activity, was directed against specific ascomycetes, and synergistic interaction with the chitin synthetase inhibitor nikkomycin inhibited growth of Aspergillus species. Microscopy studies revealed that fluorescein-labeled AFP1 strongly bound to the surface of germinated conidia and to tips of growing hyphae, causing severe alterations in cell morphogenesis that gave rise to large spherical conidia and/or swollen hyphae and to atypical branching.
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5. |
Schwarz W,
Möhrle V,
Lauer B,
Bruntner C,
( 1999 ) Molecular characterization of co-transcribed genes from Streptomyces tendae T?901 involved in the biosynthesis of the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin. PMID : 10503541 : DOI : 10.1007/pl00008637 Abstract >>
Six genes (nikA, nikB, nikD, nikE, nikF, and nikG) from Streptomyces tendae T?901 were identified by sequencing the region surrounding the nikC gene, which encodes L-lysine 2-aminotransferase, previously shown to catalyze the initial reaction in the biosynthesis of hydroxypyridylhomothreonine, the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin. These genes, together with the nikC gene, span a DNA region of 7.87 kb and are transcribed as a polycistronic mRNA in a growth-phase dependent manner. The sequences of the deduced proteins NikA and NikB exhibit significant similarity to those of acetaldehyde dehydrogenases and 4-hydroxy-2-oxovalerate aldolases, respectively, which are involved in meta-cleavage degradation of aromatic hydrocarbons. The predicted NikD gene product shows sequence similarity to monomeric sarcosine oxidases, and the deduced NikE protein belongs to the superfamily of adenylate-forming enzymes. The nikF gene and the nikG gene encode a cytochrome P450 monooxygenase and a ferredoxin, respectively. Disruption of any of the genes nikA, nikB, nikD, nikE and nikF by insertion of a kanamycin resistance cassette abolished formation of the biologically active nikkomycins I, J, X, and Z. The nikA, nikB, nikD, and nikE mutants accumulated the nucleoside moieties nikkomycins Cx and Cz. In the nikD and nikE mutants nikkomycin production (nikkomycins I, J, X, Z) could be restored by feeding with picolinic acid and hydroxypyridylhomothreonine, respectively. The nikF mutant exclusively produced novel derivatives, nikkomycins Lx and Lz, which contain pyridylhomothreonine as the peptidyl moiety. Our results indicate that the nikA, nikB, nikD, nikE, nikF, and nikG genes, in addition to nikC, function in the biosynthetic pathway leading to hydroxypyridylhomothreonine, the putative activities of each of their products are discussed.
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6. |
Carrell CJ,
Bruckner RC,
Venci D,
Zhao G,
Jorns MS,
Mathews FS,
( 2007 ) NikD, an unusual amino acid oxidase essential for nikkomycin biosynthesis: structures of closed and open forms at 1.15 and 1.90 A resolution. PMID : 17697998 : DOI : 10.1016/j.str.2007.06.010 PMC : PMC2764521 Abstract >>
NikD is an unusual amino-acid-oxidizing enzyme that contains covalently bound FAD, catalyzes a 4-electron oxidation of piperideine-2-carboxylic acid to picolinate, and plays a critical role in the biosynthesis of nikkomycin antibiotics. Crystal structures of closed and open forms of nikD, a two-domain enzyme, have been determined to resolutions of 1.15 and 1.9 A, respectively. The two forms differ by an 11 degrees rotation of the catalytic domain with respect to the FAD-binding domain. The active site is inaccessible to solvent in the closed form; an endogenous ligand, believed to be picolinate, is bound close to and parallel with the flavin ring, an orientation compatible with redox catalysis. The active site is solvent accessible in the open form, but the picolinate ligand is approximately perpendicular to the flavin ring and a tryptophan is stacked above the flavin ring. NikD also contains a mobile cation binding loop.
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7. |
Billeter M,
Kline AD,
Braun W,
Huber R,
Wüthrich K,
( 1989 ) Comparison of the high-resolution structures of the alpha-amylase inhibitor tendamistat determined by nuclear magnetic resonance in solution and by X-ray diffraction in single crystals. PMID : 2786963 : DOI : 10.1016/0022-2836(89)90575-5 Abstract >>
The three-dimensional structure of the alpha-amylase inhibitor Tendamistat determined by nuclear magnetic resonance in aqueous solution is compared with the Tendamistat crystal structure refined at 2.0 A resolution. Between the two independently obtained structures the root-mean-square distances are 1.05 A for the backbone atoms N, C alpha and C', 1.25 A for the backbone and the interior side-chains, and 1.84 A for all heavy atoms. These numbers show that the interior of the molecule is nearly identical in the two states. Near the protein surface a small number of local differences between the two structures were identified. In most surface areas the solution structure appears more disordered than the crystal structure, with the exception of Tyr15, which was not observed in the X-ray diffraction.
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8. |
Koller KP,
Riess G,
( 1989 ) Heterologous expression of the alpha-amylase inhibitor gene cloned from an amplified genomic sequence of Streptomyces tendae. PMID : 2788646 : DOI : 10.1128/jb.171.9.4953-4957.1989 PMC : PMC210302 Abstract >>
The coding region for a secreted proteinaceous inhibitor of the human alpha-amylase (tendamistat; HOE 467) was identified by using a synthetic oligonucleotide probe. The gene is part of a 37-kilobase amplified genomic sequence found in an overproducing mutant of Streptomyces tendae. After subcloning, sequence analysis revealed an open reading frame of 312 base pairs preceded by a putative ribosome-binding site. The reading frame is 30 codons longer than necessary for the mature protein. This sequence coded for an amino-terminal extension of tendamistat and shows typical features of a signal peptide. After being cloned into Streptomyces vector plasmids and transformed to the heterologous host, Streptomyces lividans TK24, the gene was expressed, and the alpha-amylase inhibitor was correctly processed and secreted into the culture medium. The amount of secreted protein was dependent on the gene dosage and on the promoter arrangement.
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9. |
Gruber K,
Macheroux P,
Oberdorfer G,
Ginj C,
( 2012 ) Structural and functional characterization of NikO, an enolpyruvyl transferase essential in nikkomycin biosynthesis. PMID : 22810238 : DOI : 10.1074/jbc.M112.352096 PMC : PMC3438971 Abstract >>
Nikkomycins are peptide-nucleoside compounds with fungicidal, acaricidal, and insecticidal properties because of their strong inhibition of chitin synthase. Thus, they are potential antibiotics especially for the treatment of immunosuppressed patients, for those undergoing chemotherapy, or after organ transplants. Although their chemical structure has been known for more than 30 years, only little is known about their complex biosynthesis. The genes encoding for proteins involved in the biosynthesis of the nucleoside moiety of nikkomycins are co-transcribed in the same operon, comprising the genes nikIJKLMNO. The gene product NikO was shown to belong to the family of enolpyruvyl transferases and to catalyze the transfer of an enolpyruvyl moiety from phosphoenolpyruvate to the 3'-hydroxyl group of UMP. Here, we report activity and inhibition studies of the wild-type enzyme and the variants C130A and D342A. The x-ray crystal structure revealed differences between NikO and its homologs. Furthermore, our studies led to conclusions concerning substrate binding and preference as well as to conclusions about inhibition/alkylation by the antibiotic fosfomycin.
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10. |
Bretschneider T,
Zocher G,
Unger M,
Scherlach K,
Stehle T,
Hertweck C,
( 2011 ) A ketosynthase homolog uses malonyl units to form esters in cervimycin biosynthesis. PMID : 22179067 : DOI : 10.1038/nchembio.746 Abstract >>
Ketosynthases produce the carbon backbones of a vast number of biologically active polyketides by catalyzing Claisen condensations of activated acyl and malonyl building blocks. Here we report that a ketosynthase homolog from Streptomyces tendae, CerJ, unexpectedly forms malonyl esters during the biosynthesis of cervimycin, a glycoside antibiotic against methicillin-resistant Staphylococcus aureus (MRSA). Deletion of cerJ yielded a substantially more active cervimycin variant lacking the malonyl side chain, and in vitro biotransformations revealed that CerJ is capable of transferring malonyl, methylmalonyl and dimethylmalonyl units onto the glycoside. According to phylogenetic analyses and elucidation of the crystal structure, CerJ is functionally and structurally positioned between the ketosynthase catalyzing Claisen condensations and acyl-ACP shuttles, and it features a noncanonical catalytic triad. Site-directed mutagenesis and structures of CerJ in complex with substrates not only allowed us to establish a model for the reaction mechanism but also provided insights into the evolution of this important subclass of the thiolase superfamily.
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11. |
( 1994 ) The nuclear-magnetic-resonance solution structure of the mutant alpha-amylase inhibitor [R19L] Tendamistat and comparison with wild-type Tendamistat. PMID : 8143730 : DOI : 10.1111/j.1432-1033.1994.tb18677.x Abstract >>
The recombinant mutant alpha-amylase inhibitor [R19L]Tendamistat, with Arg19 replaced by Leu, was prepared and its NMR solution structure determined. Based on complete sequence-specific 1H-NMR assignments, 845 nuclear Overhauser effect upper-distance constraints and 156 dihedral angle constraints were collected using two-dimensional homonuclear 1H-NMR experiments. The structure was calculated with the program DIANA, using the redundant dihedral angle constraints strategy for improved convergence. For restrained energy minimization, the programs FANTOM and AMBER were used. The wild-type NMR solution structure was similarly recalculated from the previously published input of conformational constraints [Kline, A., Braun, W. & W?thrich, K. (1988) J. Mol. Biol. 204, 675-724]. For each protein a group of 20 conformers represents a well-defined solution structure, with average root-mean-square-distance values for the backbone atoms of the individual conformers relative to the mean coordinates of 50 pm. The two structures are nearly identical to each other and to the previously published Tendamistat structures in solution and in crystals. The only significant difference is strictly localized near the single amino acid substitution in the presumed active site -Trp18-Arg(Leu)-Tyr-, i.e. Leu19 and Tyr20 are more precisely defined in the solution structure of [R19L]Tendamistat than the corresponding residues Arg19 and Tyr20 in wild-type Tendamistat.
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12. |
Möhrle V,
Roos U,
Bormann C,
( 1995 ) Identification of cellular proteins involved in nikkomycin production in Streptomyces tendae T?901. PMID : 7783626 : DOI : 10.1111/j.1365-2958.1995.tb02269.x Abstract >>
Expression of genes involved in nikkomycin production in Streptomyces tendae was investigated by two-dimensional gel electrophoresis of cellular proteins. Ten gene products (P1-P10) were identified that were synthesized when nikkomycin was produced; these proteins were not detected in non-producing mutants. N-terminal sequences of six of the 10 proteins were obtained by microsequencing of protein spots excised from preparative two-dimensional gels. Protein P8 was identified as L-histidine amino-transferase (HisAT), which has been previously correlated with nikkomycin production. By using oligonucleotide probes deduced from the N-terminal sequences of protein P2 and P6, we isolated an 8 kb BamHI fragment and a 6.5 kb PvuII fragment, respectively, from the genome of Streptomyces tendae T?901. Restriction analyses revealed that both fragments overlapped within a region of 1.5 kb. Mapping of the oligonucleotide probe hybridizing sites indicated that the genes encoding protein P2 and P6 are closely spaced on the 8 kb BamHI fragment, and the latter is located on the overlapping region. DNA sequence analysis revealed that proteins P1 and P2 are encoded by a single gene, orfP1, that is translated at two initiation codons. The orfP1 gene was interrupted by homologous recombination using the integrating vector pWHM3. The gene-disrupted transformants did not produce nikkomycin, indicating that proteins P1 and P2 are essential for nikkomycin production. The data presented show that reverse genetics was successfully used to isolate genes involved in nikkomycin production.
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13. |
Aschauer H,
Vértesy L,
Nesemann G,
Braunitzer G,
( 1983 ) [The primary structure of the alpha-amylase inhibitor Hoe 467A from Streptomyces tendae 4158. A new class of inhibitors]. PMID : 6605909 : Abstract >>
The native or modified alpha-amylase inhibitor Hoe 467A - isolated from the culture medium of Streptomyces tendae 4158 - and overlapping peptides were degraded by the automatic Edman technique. The oxidized or aminoethylated or oxidized and maleoylated inhibitor was digested with trypsin and the native inhibitor with pepsin. Further digestion with Staphylococcus aureus proteinase was also carried out. After peptic digestion two cystin peptides were isolated, which allowed the establishment of the disulfide bonds. The alpha-amylase inhibitor is a polypeptid consisting of 74 amino-acid residues with a molecular mass of 7958 Da. The inhibitor is composed of all naturally occurring amino acids except methionine and phenylalanine and shows no sequence homology to known inhibitors. The clinical and pharmacological importance in respect to the inhibitors ability for inactivation of human salivary and pancreatic alpha-amylase is discussed. Especially the proteinase resistance of the inhibitor enables a clinical application in human (e.g. Diabetes mellitus) per os.
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14. |
( 1995 ) The crystal structure of porcine pancreatic alpha-amylase in complex with the microbial inhibitor Tendamistat. PMID : 7897663 : DOI : 10.1006/jmbi.1994.0125 Abstract >>
The crystal structure of the complex formed between the 498 amino acid residue porcine pancreatic alpha-amylase (PPA) and the 74 amino acid residue inhibitor Tendamistat secreted from Streptomyces tendae, has been determined by multiple isomorphous replacement in a crystal of space group P6(5)22 (a = b = 77.7 A, c = 359.5 A). The model has been refined to an R-factor of 0.194 by Powell minimization applying strong energy constraints based on 17,964 independent reflections in the 7 to 2.5 A resolution range, and obeys standard geometry within 0.011 A in bond lengths and 1.78 degrees in bond angles. The final model consists of all 496 amino acid residues of PPA, 71 amino acid residues of Tendamistat (without the three N-terminal residues), one calcium ion, one chloride ion and 167 water molecules. PPA exhibits the same topological fold in the complex as the uncomplexed PPA recently published by others. About 30% of the water-accessible surface of Tendamistat is in contact with PPA. Four segments of the polypeptide chain, with a total of 15 amino acid residues, are involved in the binding. One segment containing the staggered side-chains of the triplet Trp18, Arg19, Tyr20, typical for this class of inhibitors, binds into the catalytic site. The other segments fill out the groove in the PPA molecule, which also binds the carbohydrate inhibitor acarbose and is assumed to be the substrate-binding region. This extended interaction between Tendamistat and alpha-amylase explains the very high inhibition constant of about 9 x 10(-12) M.
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15. |
Vértesy L,
Oeding V,
Bender R,
Zepf K,
Nesemann G,
( 1984 ) Tendamistat (HOE 467), a tight-binding alpha-amylase inhibitor from Streptomyces tendae 4158. Isolation, biochemical properties. PMID : 6611258 : DOI : 10.1111/j.1432-1033.1984.tb08221.x Abstract >>
Culture fluids of Streptomyces tendae 4158 (ATCC 31210) contain a new kind of polypeptide alpha-amylase inhibitor, tendamistat (HOE 467). Several methods of isolating this inhibitor are described, including two rapid crystallisation methods, which produce homogeneous material. A characteristic of tendamistat is its tight-binding, pH-independent inhibition kinetics and the specific inhibition of the mammalian alpha-amylase form a stoichiometric 1:1 complex, which cannot be separated into its individual components by sodium dodecyl sulphate or molecular sieve chromatography. Studies of the mode of action reveal that the alpha-amylase-inhibiting activity is linked to the intact disulphide bridges of the inhibitor. It is assumed that the multipoint protein-protein bond exists between the enzyme and tendamistat. It is shown that extracellular tendamistat inhibits amylase formed by streptomyces. We therefore assume a regulatory function in the microorganism. By-products of tendamistat, which possess similar enzyme-inhibiting properties, are also described.
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16. |
Kline AD,
Braun W,
Wüthrich K,
( 1988 ) Determination of the complete three-dimensional structure of the alpha-amylase inhibitor tendamistat in aqueous solution by nuclear magnetic resonance and distance geometry. PMID : 3265733 : DOI : 10.1016/0022-2836(88)90364-6 Abstract >>
The complete three-dimensional structure of the alpha-amylase inhibitor Tendamistat in aqueous solution was determined by 1H nuclear magnetic resonance and distance geometry calculations using the program DISMAN. Compared to an earlier, preliminary determination of the polypeptide backbone conformation, stereo-specific assignments were obtained for 41 of the 89 prochiral groups in the protein, and a much more extensive set of experimental constraints was collected, including 842 distance constraints from nuclear Overhauser effects and over 100 supplementary constraints from spin-spin coupling constants and the identification of intramolecular hydrogen bonds. The complete protein molecule, including the amino acid side-chains is characterized by a group of nine structures corresponding to the results of the nine DISMAN calculations with minimal residual error functions. The average of the pairwise minimal root-mean-square distances among these nine structures is 0.85 A for the polypeptide backbone, and 1.52 A for all the heavy atoms. The procedures used for the structure determination are described and a detailed analysis is presented of correlations between the experimental input data and the precision of the structure determination.
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17. |
Pflugrath JW,
Wiegand G,
Huber R,
Vértesy L,
( 1986 ) Crystal structure determination, refinement and the molecular model of the alpha-amylase inhibitor Hoe-467A. PMID : 3489104 : DOI : 10.1016/0022-2836(86)90520-6 Abstract >>
The crystal and molecular structure of the alpha-amylase inhibitor Hoe-467A has been determined and refined at high resolution. The polypeptide chain is folded in two triple-stranded sheets, which form a barrel. The topology of folding is as found in the immunoglobulin domains. The amino acid triplet Trp18-Arg19-Tyr20 has an exceptional conformation and position in the molecule and is possibly involved in inhibitory activity.
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18. |
Kline AD,
Wüthrich K,
( 1985 ) Secondary structure of the alpha-amylase polypeptide inhibitor tendamistat from Streptomyces tendae determined in solution by 1H nuclear magnetic resonance. PMID : 3874968 : DOI : 10.1016/0022-2836(85)90018-x Abstract >>
Complete sequence-specific 1H nuclear magnetic resonance assignments were obtained for the backbone hydrogen atoms in Tendamistat, a protein with 74 residues. From NOESY observation of 1H-1H short distance constraints, measurements of the spin-spin couplings 3JHN alpha and a qualitative identification of slowly exchanging amide protons, two antiparallel beta-sheets containing three and four strands, respectively, were identified. The peptide segments outside the beta-sheets do not form regular secondary structure. Preliminary data were obtained on the relative spatial arrangements of the two beta-sheets.
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19. |
Hu Y,
Sun F,
Liu W,
( 2018 ) The heat shock protein 70 gene as a new alternative molecular marker for the taxonomic identification of Streptomyces strains. PMID : 30203150 : DOI : 10.1186/s13568-018-0674-4 PMC : PMC6134474 Abstract >>
With the developments in taxonomy, the classically used highly conserved 16S rRNA molecular marker has shown some disadvantages among closely related species. For further taxonomic studies of the prokaryotes, specific PCR primers were designed from two conserved regions in the amino acid sequences of the 70-kDa heat shock protein sourced from 20 different genera in actinomycetes. These were used for the amplification of the hsp70 genes in 16 Streptomyces strains. Then, we investigated the phylogenetic relationships among these Streptomyces strains and compared the tree topology based on the hsp70 gene with those based on the previously used markers (16S rRNA and gyrB). To our knowledge, this is the first use of the hsp70 gene as a molecular marker for the taxonomic identification of Streptomyces.
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20. |
( 1998 ) The Streptomyces tendae T?901 L-lysine 2-aminotransferase catalyzes the initial reaction in nikkomycin D biosynthesis. PMID : 9660190 : DOI : 10.1046/j.1432-1327.1998.2540347.x Abstract >>
Protein P8 was previously identified as a putative nikkomycin biosynthesis protein. The gene (nikC) encoding protein P8 was cloned from the Streptomyces tendae T?901 nikkomycin gene cluster and sequenced. The nikC gene was inactivated by inserting a kanamycin resistance cassette; the mutant did not produce the biologically active nikkomycins I, J, X, and Z, but accumulated the nucleoside moieties nikkomycins C(X) and C(Z). The mutant was complemented to nikkomycin production (I, J, X, Z) by nikC expressed from the mel promoter of the vector pIJ702. Furthermore, the nikkomycin-negative phenotype was reversed by the addition of picolinic acid, a precursor of the peptidyl moiety of nikkomycins (nikkomycin D), into the culture medium. The nikC gene was expressed in Escherichia coli and identified and characterized at the enzyme level. NikC encodes an L-lysine 2-aminotransferase, and the activity was exclusively detected in nikkomycin producers and its presence correlated to nikkomycin production. The nikC-inactivated mutant grew with L-lysine as sole source of nitrogen and carbon, indicating that L-lysine 2-aminotransferase is not required for lysine catabolism. Our results identified the nikC-encoded L-lysine 2-aminotransferase as the nikkomycin biosynthetic enzyme that catalyzes the initial reaction in nikkomycin D biosynthesis. The NikC protein belongs to a novel family of pyridoxamine or pyridoxal-phosphate-dependent dehydrases and aminotransferases, some of which are involved in dideoxy- and deoxyaminosugar biosynthesis.
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