-
1
المؤلفون: Jinghua Xue, Lei Yao, Chen Wang, Deng Wangqiu, Hanxiang Li, Liangxiong Xu, Xiaoyi Wei, Ping Wu
المصدر: The Journal of Antibiotics. 71:927-938
مصطلحات موضوعية: Models, Molecular, Pharmacology, Protein Conformation, 010405 organic chemistry, Stereochemistry, Chemistry, Ab initio, Sequence alignment, 01 natural sciences, 0104 chemical sciences, Acremonium, 010404 medicinal & biomolecular chemistry, Residue (chemistry), Protein structure, Drug Discovery, Amphiphile, Density functional theory, Amino Acid Sequence, Peptides, Two-dimensional nuclear magnetic resonance spectroscopy, Peptide sequence
الوصف: Four new peptaibiotics, acremotins A–D (1–4) featuring three α,α-dialkylated amino acid–imino acid motifs and an unreduced C-terminal residue, along with the known peptaibiotic XR586 (5) were isolated from the solid cultures of the soil-derived fungus Acremonium persicinum SC0105. Their primary structures were characterized by detailed analysis of the HRESIMS/MS fragmentation pattern combined with comprehensive interpretation of the 1D and 2D NMR spectroscopic data. The absolute configurations of amino acid residues were determined by the advanced Marfey’s method. Sequence alignment result shows that 1–4 are closely related to zervamicin IIB and emerimicin IIA, thus belong to peptaibiotic subfamily-3 (SF3). The three-dimensional (3D) structure of 4 was established by theoretical conformational analysis using the ab initio density functional theory (DFT) method, which, together with the CD spectrum, indicated an amphiphilic and helical structure for 4. 1–5 actively inhibited the growth of gram-positive bacterial pathogens, and amongst them 4 was the most potent compound showing MIC of 12.5 and 6.25 µg/ml against S. aureu and MRSA strains, respectively. 1–5 were also cytotoxic against three human cancer cell lines with IC50 ranging from 1.2 to 21.6 μM.
-
2
المؤلفون: Thomas Degenkolb, Stefan Fox, Hans Brückner
المصدر: Chemistrybiodiversity. 16(9)
مصطلحات موضوعية: chemistry.chemical_classification, Spectrometry, Mass, Electrospray Ionization, Stereochemistry, Bioengineering, Stereoisomerism, General Chemistry, General Medicine, Mass spectrometry, Biochemistry, Amino acid, Anti-Bacterial Agents, Acremonium, chemistry.chemical_compound, chemistry, Isovaline, Acremonium crotocinigenum, Molecular Medicine, 1-aminocyclopropanecarboxylic acid, Amino Acid Sequence, Peptides, Molecular Biology, Chromatography, High Pressure Liquid
-
3
المؤلفون: Han Shu, Youjia Hu, Guihua Gong, Zhang Wei, Pan Jie, Liping Xie, Song Zhihui
المصدر: Biochemistry. Biokhimiia. 82(7)
مصطلحات موضوعية: 0301 basic medicine, Magnetic Resonance Spectroscopy, Pyrimidine, Stereochemistry, Glycine, Ligands, Biochemistry, Cofactor, Mass Spectrometry, Fungal Proteins, 03 medical and health sciences, chemistry.chemical_compound, Biosynthesis, Amino Acid Sequence, Thiazole, Chromatography, High Pressure Liquid, chemistry.chemical_classification, biology, Chemistry, General Medicine, NAD, Recombinant Proteins, Acremonium, Adenosine Diphosphate, Thiazoles, 030104 developmental biology, Enzyme, biology.protein, Mutagenesis, Site-Directed, Thiamine, NAD+ kinase, Thiamine Pyrophosphate, Sequence Alignment, Thiamine pyrophosphate
الوصف: Thiamine pyrophosphate is an essential coenzyme in all organisms. Its biosynthesis involves independent syntheses of the precursors, pyrimidine and thiazole, which are then coupled. In our previous study with overexpressed and silent mutants of ActhiS (thiazole biosynthesis enzyme from Acremonium chrysogenum), we found that the enzyme level correlated with intracellular thiamine content in A. chrysogenum. However, the exact structure and function of ActhiS remain unclear. In this study, the enzyme-bound ligand was characterized as the ADP adduct of 5-(2-hydroxyethyl)-4-methylthiazole-2-carboxylic acid (ADT) using HPLC and 1H NMR. The ligand-free ActhiS expressed in M9 minimal medium catalyzed conversion of NAD+ and glycine to ADT in the presence of iron. Furthermore, the C217 residue was identified as the sulfur donor for the thiazole moiety. These observations confirm that ActhiS is a thiazole biosynthesis enzyme in A. chrysogenum, and it serves as a sulfur source for the thiazole moiety.
-
4
المؤلفون: Meng-Hwan Lee, Wen-Lin Lai, Shwu-Huey Liaw, Shuen-Fuh Lin, Cheng-Sheng Hsu, Ying-Chieh Tsai
المصدر: Applied and Environmental Microbiology. 71:8881-8887
مصطلحات موضوعية: Stereochemistry, Molecular Sequence Data, Flavoprotein, Flavin group, Applied Microbiology and Biotechnology, Pichia pastoris, chemistry.chemical_compound, Amino Acid Sequence, Cytokinin dehydrogenase, Enzymology and Protein Engineering, DNA Primers, Alanine, Flavin adenine dinucleotide, Base Sequence, Sequence Homology, Amino Acid, Ecology, biology, Acremonium strictum, biology.organism_classification, TATA Box, Peptide Fragments, Recombinant Proteins, Acremonium, Alcohol Oxidoreductases, Amino Acid Substitution, Biochemistry, chemistry, Spectrophotometry, biology.protein, Sequence Alignment, Food Science, Biotechnology, Cysteine
الوصف: Glucooligosaccharide oxidase from Acremonium strictum was screened for potential applications in oligosaccharide acid production and carbohydrate detection. This protein is a unique covalent flavoenzyme which catalyzes the oxidation of a variety of carbohydrates with high selectivity for cello- and maltooligosaccharides. Kinetic measurements suggested that this enzyme possesses an open carbohydrate-binding groove, which is mainly composed of two glucosyl-binding subsites. The encoding gene was subsequently cloned, and one intron was detected in the genomic DNA. Large amounts of active enzymes were expressed in Pichia pastoris , with a yield of 300 mg per liter medium. The protein was predicted to share structural homology with plant cytokinin dehydrogenase and related flavoproteins that share a conserved flavin adenine dinucleotide (FAD)-binding domain. The closest sequence matches are those of plant berberine bridge enzyme-like proteins, particularly the characteristic flavinylation site. Unexpectedly, mutation of the putative FAD-attaching residue, H70, to alanine, serine, cysteine, and tyrosine did not abolish the covalent FAD linkage and had little effect on the K m . Instead, the variants displayed k cat values that were 50- to 600-fold lower, indicating that H70 is crucial for efficient redox catalysis, perhaps through modulation of the oxidative power of the flavin.
URL الوصول: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::28850264cbb5912300c7804cdc933624
https://doi.org/10.1128/aem.71.12.8881-8887.2005 -
5
المؤلفون: Wendy J. Sobey, Jack E. Baldwin, Christopher J. Schofield, Chia-Yang Shiau, Michael F. Byford
مصطلحات موضوعية: β-Lactam, Magnetic Resonance Spectroscopy, Stereochemistry, Electrospray ionization, Molecular Sequence Data, Biophysics, Peptide, Tripeptide, Biosynthesis, Biochemistry, Mass Spectrometry, chemistry.chemical_compound, Structural Biology, Valine, Serine, Genetics, Peptide bond, Amino Acid Sequence, Peptide Synthases, Molecular Biology, Epimerisation, chemistry.chemical_classification, Synthetase, Stereoisomerism, Dipeptides, Cell Biology, Amino acid, Models, Chemical, chemistry, Cephalosporium acremonium, Cysteine
الوصف: delta-L-(alpha-Aminoadipoyl)-L-cysteinyl-D-valine (ACV) synthetase catalyses the formation of the common precursor tripeptide of both the penicillin and cephalosporin antibiotics from the L-enantiomers of its constituent amino acids. Replacement of cysteine with L-O-methylserine in preparative-scale incubations led to the isolation of both L-O-methylserinyl-L-valine and L-O-methylserinyl-D-valine dipeptides. The dipeptides were characterized with the aid of authentic synthetic standards by both 1H NMR and electrospray ionization MS. A revised mechanism for ACV biosynthesis involving formation of the cysteinyl-valine peptide bond before the epimerisation of valine and subsequent condensation with the delta-carboxyl of L-alpha-aminoadipate is therefore proposed.
URL الوصول: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::88debaa6b9783d5a04bc0b37c9d4e3d9
https://doi.org/10.1016/0014-5793(94)01320-z -
6
المؤلفون: Charles M. H. Hensgens, Sarah J. Lipscomb, Jack E. Baldwin, Kirsty S. Hewitson, Matthew D. Lloyd, Christopher J. Schofield
المصدر: Journal of Biological Chemistry. 279:15420-15426
مصطلحات موضوعية: Models, Molecular, Penicillin binding proteins, Stereochemistry, Iron, Molecular Sequence Data, Mutant, Streptomyces clavuligerus, Penicillins, Biochemistry, Substrate Specificity, Hydroxylation, chemistry.chemical_compound, Methionine, Penicillin-Binding Proteins, Point Mutation, Amino Acid Sequence, Cloning, Molecular, Intramolecular Transferases, Molecular Biology, chemistry.chemical_classification, Binding Sites, Sequence Homology, Amino Acid, biology, Deacetoxycephalosporin-C synthase, Tryptophan, Active site, Cell Biology, Cephalosporin C, biology.organism_classification, Streptomyces, Cephalosporins, Protein Structure, Tertiary, Acremonium, Oxygen, Kinetics, Enzyme, Models, Chemical, chemistry, Mutation, Mutagenesis, Site-Directed, Oxygenases, biology.protein, Asparagine, Protein Binding
الوصف: Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutarate-dependent oxygenase involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium. It catalyzes two oxidative reactions, oxidative ring-expansion of penicillin N to deacetoxycephalosporin C, and hydroxylation of the latter to give deacetylcephalosporin C. The enzyme is closely related to deacetoxycephalosporin C synthase (DAOCS) and DACS from Streptomyces clavuligerus, which selectively catalyze ring-expansion or hydroxylation reactions, respectively. In this study, structural models based on DAOCS coupled with site-directed mutagenesis were used to identify residues within DAOC/DACS that are responsible for controlling substrate and reaction selectivity. The M306I mutation abolished hydroxylation of deacetylcephalosporin C, whereas the W82A mutant reduced ring-expansion of penicillin G (an "unnatural" substrate). Truncation of the C terminus of DAOC/DACS to residue 310 (Delta310 mutant) enhanced ring-expansion of penicillin G by approximately 2-fold. A double mutant, Delta310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS. The Delta310/N305L/M306I triple mutant selectively catalyzed ring-expansion of penicillin G and had improved kinetic parameters (K(m) = 2.00 +/- 0.47 compared with 6.02 +/- 0.97 mm for the wild-type enzyme). This work demonstrates that a single amino acid residue side chain within the DAOC/DACS active site can control whether the enzyme catalyzes ring-expansion, hydroxylation, or both reactions. The catalytic efficiency of mutant enzymes can be improved by combining active site mutations with other modifications including C-terminal truncation and modification of Asn-305.
URL الوصول: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::734c6c04b106b5680015ac23ef2e716e
https://doi.org/10.1074/jbc.m313928200 -
7
المؤلفون: Norihiro Tsukagoshi, Sachiko Kajita, Masashi Kato, Koichi Banno, Masayasu Sugino, Takashi Yamane, Tetsuo Kobayashi, Tsuyoshi Shirai
المصدر: Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1596:36-46
مصطلحات موضوعية: Models, Molecular, Stereochemistry, Molecular Sequence Data, Mutant, Biophysics, Biochemistry, Catalysis, Fungal Proteins, Electron transfer, chemistry.chemical_compound, Structural Biology, Enzyme Stability, Amino Acid Sequence, Site-directed mutagenesis, Molecular Biology, Thermostability, chemistry.chemical_classification, Binding Sites, biology, Acremonium, Hydrogen-Ion Concentration, biology.organism_classification, Amino acid, Enzyme, chemistry, Mutagenesis, Site-Directed, Ascorbate Oxidase, Azide, Sequence Alignment, Plasmids
الوصف: A gene encoding a thermostable Acremonium ascorbate oxidase (ASOM) was randomly mutated to generate mutant enzymes with altered pH optima. One of the mutants, which exhibited a significantly higher activity in the pH range 4.5–7 compared to ASOM, had a Gln183Arg substitution in the region corresponding to SBR1, one of the substrate binding regions of the zucchini enzyme. The other mutant with almost the same pH profile as Gln183Arg had a Thr527Ala substitution near the type 3 copper center and became more sensitive to azide than ASOM. Site-directed mutagenesis in the substrate binding regions with reference to the amino acid sequences of plant enzymes led to isolation of mutants shifted upward in the pH optimum; Val193Pro and Val193Pro/Pro190Ile increased the pH optimum by 1 and 0.5 units, respectively, while retaining the near-wild-type thermostability and azide sensitivity. The homology model of ASOM constructed from the zucchini enzyme coordinates suggested that replacement of Val193 by Pro could disturb the ion pair networks among Arg309, Glu192, Arg194 and Glu311. This perturbation could affect either the molecular recognition between the substrate and ASOM or the electron transfer from the substrate to the type 1 copper center, leading to the alkaline shift of the catalytic activity of the mutant enzyme. The other mutations, Val193Pro/Pro190Ile, could also induce similar structural perturbations involving the ion pair networks.
-
8
المؤلفون: Tiow-Suan Sim, Paxton Loke
المصدر: Biochemical and Biophysical Research Communications. 252:472-475
مصطلحات موضوعية: Stereochemistry, Glutamine, Molecular Sequence Data, Biophysics, Isopenicillin N synthase, Ligands, Biochemistry, Aspergillus nidulans, Catalytic Domain, Amino Acid Sequence, Site-directed mutagenesis, Molecular Biology, DNA Primers, chemistry.chemical_classification, Base Sequence, Sequence Homology, Amino Acid, biology, Ligand, Acremonium, Cell Biology, biology.organism_classification, Amino acid, Isoenzymes, Enzyme, chemistry, Mutagenesis, Site-Directed, biology.protein, Oxidoreductases
الوصف: Isopenicillin N synthase (IPNS), an important enzyme in the beta-lactam antibiotic biosynthetic pathway, is responsible for the catalytic conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. Three catalytic ligands essential for IPNS activity have already been determined. Based on an Aspergillus nidulans IPNS crystal structure, the probable involvement of a fourth amino acid as a catalytic ligand was previously revealed. To continue the search for the fourth catalytic ligand, we report investigations on whether or not glutamines play a role in the catalytic action of Cephalosporium acremonium IPNS (cIPNS). Three glutamine residues were targeted for modification based on the previous revelation of one (Q337) via crystal structure coordinates, the conservation of one (Q234) in isozyme alignment and the proximity of one (Q227) to the catalytic centre. Analysis of the biotransformed mutant enzymes showed retention of activity, thereby rejecting the involvement of a possible glutamine as a catalytic ligand in cIPNS catalysis.
-
9
المؤلفون: Jack E. Baldwin, Karl Harlos, Inger Andersson, Ian J. Clifton, Geoffrey J. Barton, Peter L. Roach, Christopher J. Schofield, Janos Hajdu, Vilmos Fülöp
المصدر: Scopus-Elsevier
مصطلحات موضوعية: Protein Conformation, Stereochemistry, Molecular Sequence Data, Thiazolidine, Isopenicillin N synthase, Tripeptide, Crystallography, X-Ray, Aspergillus nidulans, Catalysis, chemistry.chemical_compound, Protein structure, Computer Graphics, polycyclic compounds, Amino Acid Sequence, Cloning, Molecular, Antibacterial agent, Manganese, Binding Sites, Multidisciplinary, Sequence Homology, Amino Acid, biology, Deacetoxycephalosporin-C synthase, Bicyclic molecule, Active site, Recombinant Proteins, Acremonium, chemistry, biology.protein, Oxidoreductases
الوصف: Penicillin antibiotics are all produced from fermentation-derived penicillins because their chemical synthesis is not commercially viable. The key step in penicillin biosynthesis, in which both the beta-lactam and thiazolidine rings of the nucleus are created, is mediated by isopenicillin N synthase (IPNS), which binds ferrous iron and uses dioxygen as a cosubstrate. In a unique enzymatic step, with no chemical precedent, IPNS catalyses the transfer of four hydrogen atoms from its tripeptide substrate to dioxygen forming, in a single reaction, the complete bicyclic nucleus of the penicillins. We now report the structure of IPNS complexed with manganese, which reveals the active site is unusually buried within a 'jelly-roll' motif and lined by hydrophobic residues, and suggest how this structure permits the process of penicillin formation. Sequence analyses indicate IPNS, 1-aminocyclopropane-1-carboxylic acid oxidase and many of the 2-oxo-acid-dependent oxygenases contain a conserved jelly-roll motif, forming a new structural family of enzymes.
-
10
المؤلفون: Paxton Loke, Tiow-Suan Sim
المصدر: FEMS microbiology letters. 179(2)
مصطلحات موضوعية: Stereochemistry, Molecular Sequence Data, Isopenicillin N synthase, Biology, Arginine, Microbiology, Protein Structure, Secondary, Substrate Specificity, Serine, Genetics, Amino Acid Sequence, Binding site, Site-directed mutagenesis, Molecular Biology, Alanine, chemistry.chemical_classification, Acremonium strictum, biology.organism_classification, Amino acid, Acremonium, Biochemistry, chemistry, Mutation, biology.protein, Mutagenesis, Site-Directed, Leucine, Oxidoreductases
الوصف: Isopenicillin N synthase (IPNS) catalyses a key step in the penicillin and cephalosporin biosynthetic pathway which involves the oxidative cyclisation of the acyclic peptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N. Based on crystallographic evidence from the Aspergillus nidulans IPNS crystal structure complexed with the substrate ACV (Roach et al. (1997) Nature 387, 827-830), we were able to provide mutational evidence for the critical involvement of the conserved R-X-S motif in ACV binding in IPNS. The crystal structure further implicated arginine-87 in the binding of the aminoadipyl portion of ACV. Thus, in this study, the site-directed mutagenesis of the corresponding arginine-89 in Cephalosporium acremonium IPNS (cIPNS) was performed to ascertain its role in cIPNS. Alteration of arginine-89 to five amino acids from different amino acid groups, namely lysine, serine, alanine, aspartate and leucine, was performed and no activity was detected in all the mutants obtained when enzyme bioassays were performed. Furthermore, the solubility of the mutants was considerably lower than the wild-type cIPNS after expression at 37 degrees C, but could be recovered when the expression temperature was lowered to 25 degrees C. This suggests that arginine-89 could be critical for the activity of cIPNS due to its involvement in ACV binding and the solubility of wild-type enzyme.
إعادة تعيين المنقحات
عرض المنقحات (%%عد%%)
Find this article in full text from Springer Verlag.