scholarly journals Acetylacetone-cleaving enzyme Dke1: a novel C-C-bond-cleaving enzyme from Acinetobacter johnsonii

2003 ◽  
Vol 369 (3) ◽  
pp. 573-581 ◽  
Author(s):  
Grit D. STRAGANZ ◽  
Anton GLIEDER ◽  
Lothar BRECKER ◽  
Douglas W. RIBBONS ◽  
Walter STEINER
Keyword(s):  
Metabolic Pathway ◽  
Metabolic Pathways ◽  
Active Protein ◽  
Acinetobacter Johnsonii ◽  
High Affinity ◽  
Gene Coding ◽  
High Yields ◽  
Relationship Of ◽  
First Time

The toxicity of acetylacetone has been demonstrated in various studies. Little is known, however, about metabolic pathways for its detoxification or mineralization. Data presented here describe for the first time the microbial degradation of acetylacetone and the characterization of a novel enzyme that initiates the metabolic pathway. From an Acinetobacter johnsonii strain that grew with acetylacetone as the sole carbon source, an inducible acetylacetone-cleaving enzyme was purified to homogeneity. The corresponding gene, coding for a 153 amino acid sequence that does not show any significant relationship to other known protein sequences, was cloned and overexpressed in Escherichia coli and gave high yields of active enzyme. The enzyme cleaves acetylacetone to equimolar amounts of methylglyoxal and acetate, consuming one equivalent of molecular oxygen. No exogenous cofactor is required, but Fe2+ is bound to the active protein and essential for its catalytic activity. The enzyme has a high affinity for acetylacetone with a Km of 9.1μM and a kcat of 8.5s-1. A metabolic pathway for acetylacetone degradation and the putative relationship of this novel enzyme to previously described dioxygenases are discussed.

scholarly journals Characterization of Each St and Y Genome Chromosome of Roegneria grandis Based on Newly Developed FISH Markers

2021 ◽  
pp. 213-222
Author(s):  
Dandan Wu ◽  
Xiaoxia Zhu ◽  
Lu Tan ◽  
Haiqin Zhang ◽  
Lina Sha ◽  
...  
Keyword(s):  
Structural Changes ◽  
Distribution Patterns ◽  
Proximal Region ◽  
Genome Chromosome ◽  
High Affinity ◽  
Complete Set ◽  
Genome Analyses ◽  
First Time ◽  
Homoeologous Chromosomes

The genera of the tribe Triticeae (family Poaceae), constituting many economically important plants with abundant genetic resources, carry genomes such as St, H, P, and Y. The genome symbol of <i>Roegneria</i> C. Koch (Triticeae) is StY. The St and Y genomes are crucial in Triticeae, and tetraploid StY species participate extensively in polyploid speciation. Characterization of St and Y nonhomologous chromosomes in StY-genome species could help understand variation in the chromosome structure and differentiation of StY-containing species. However, the high genetic affinity between St and Y genome and the deficiency of a complete set of StY nonhomologous probes limit the identification of St and Y genomes and variation of chromosome structures among <i>Roegneria</i> species. We aimed to identify St- and Y-enhanced repeat clusters and to study whether homoeologous chromosomes between St and Y genomes could be accurately identified due to high affinity. We employed comparative genome analyses to identify St- and Y-enhanced repeat clusters and generated a FISH-based karyotype of <i>R. grandis</i> (Keng), one of the taxonomically controversial StY species, for the first time. We explored 4 novel repeat clusters (StY_34, StY_107, StY_90, and StY_93), which could specifically identify individual St and Y nonhomologous chromosomes. The clusters StY_107 and StY_90 could identify St and Y addition/substitution chromosomes against common wheat genetic backgrounds. The chromosomes V_St, VII_St, I_Y, V_Y, and VII_Y displayed similar probe distribution patterns in the proximal region, indicating that the high affinity between St and Y genome might result from chromosome rearrangements or transposable element insertion among V_St/Y, VII_St/Y, and I_Y chromosomes during allopolyploidization. Our results can be used to employ FISH further to uncover the precise karyotype based on colinearity of Triticeae species by using the wheat karyotype as reference, to analyze diverse populations of the same species to understand the intraspecific structural changes, and to generate the karyotype of different StY-containing species to understand the interspecific chromosome variation.

scholarly journals The region around residue 115 of human bactericidal/permeability-increasing protein is not involved in lipopolysaccharide binding or bactericidal activity. Chemical synthesis and expression of a gene coding for the active domain and characterization of recombinant proteins

1994 ◽  
Vol 298 (3) ◽  
pp. 711-718 ◽  
Author(s):  
S Y Qi ◽  
Y Li ◽  
C D O'Connor
Keyword(s):  
Bactericidal Activity ◽  
Polymorphonuclear Leucocytes ◽  
Recombinant Polypeptide ◽  
Outer Membranes ◽  
High Affinity ◽  
Gene Coding ◽  
High Concentrations ◽  
Derivatives Of ◽  
Lipopolysaccharide Binding

Bactericidal/permeability-increasing protein (BPI) is a potent antimicrobial agent produced by polymorphonuclear leucocytes that specifically interacts with and kills Gram-negative bacteria. An 825 bp gene determining the bactericidal N-terminal domain of human BPI was chemically synthesized and expressed as inclusion bodies in Escherichia coli. The recombinant polypeptide, BPI', was solubilized and conditions under which it folded to give the active protein were determined. Folding was critically dependent on the urea and salt concentrations as well as the pH. BPI' bound with high affinity to Salmonella typhimurium cells (apparent Kd = 36 nM), permeabilized their outer membranes to actinomycin D, specifically activated a synovial fluid phospholipase A2 and showed potent bactericidal activity. In contrast with the native protein, however, it could not be efficiently released from the cell surface by the addition of high concentrations of Mg2+ ions. Pre-incubation of the protein with lipopolysaccharide or trypsin prevented cytotoxicity. However, boiling BPI' immediately before its addition to cells did not block its bactericidal activity, suggesting that it may be able to function even when presented to cells in an unfolded form. A BPI' derivative, containing a 13-residue foreign antigenic determinant genetically inserted between Ala115 and Asp116, was also produced. The derivative was functional in the above assays and bound with high affinity to S. typhimurium (apparent Kd = 74 nM). These results imply that the region defined by these residues is not involved in the lipopolysaccharide-binding or bactericidal activities of BPI. The availability of functional, nonglycosylated recombinant derivatives of BPI should greatly aid detailed studies on its structure, interactions with lipopolysaccharide and mechanism of action.

scholarly journals Molecular Identification and Characterization of an Essential Pyruvate Transporter from Trypanosoma brucei

2013 ◽  
Vol 288 (20) ◽  
pp. 14428-14437 ◽  
Author(s):  
Marco A. Sanchez
Keyword(s):  
Trypanosoma Brucei ◽  
Drug Target ◽  
Life Cycle Stage ◽  
Bloodstream Form ◽  
Potential Drug Target ◽  
High Affinity ◽  
Physiological Relevance ◽  
First Time ◽  
Identification And Characterization

Pyruvate export is an essential physiological process for the bloodstream form of Trypanosoma brucei as the parasite would otherwise accumulate this end product of glucose metabolism to toxic levels. In the studies reported here, genetic complementation in Saccharomyces cerevisiae has been employed to identify a gene (TbPT0) that encodes this vital pyruvate transporter from T. brucei. Expression of TbPT0 in S. cerevisiae reveals that TbPT0 is a high affinity pyruvate transporter. TbPT0 belongs to a clustered multigene family consisting of five members, whose expression is up-regulated in the bloodstream form. Interestingly, TbPT family permeases are related to polytopic proteins from plants but not to characterized monocarboxylate transporters from mammals. Remarkably, inhibition of the TbPT gene family expression in bloodstream parasites by RNAi is lethal, confirming the physiological relevance of these transporters. The discovery of TbPT0 reveals for the first time the identity of the essential pyruvate transporter and provides a potential drug target against the mammalian life cycle stage of T. brucei.

scholarly journals Optimized methods for IL-17A refolding and anti-IL17A Fab production for co-crystallization with small molecules

BioTechniques ◽  
2020 ◽  
Vol 69 (1) ◽  
pp. 70-76
Author(s):  
Xiaoyun Meng ◽  
Lanjun Zhang ◽  
Hong Wei ◽  
Furong Li ◽  
Lihua Hu ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Small Molecules ◽  
Small Molecule ◽  
Yield Enhancement ◽  
High Affinity ◽  
Interleukin 17A ◽  
Affinity Peptide ◽  
First Time ◽  
Essential Prerequisite

Refolding of human interleukin 17A (IL-17A) has been reported; however, the key refolding protocol was not robust enough to deliver consistent results and to be easily scaled up for crystallization. Here we report an optimized refolding method for IL-17A. Although co-crystal structures of IL-17A with ligands have been obtained with a high-affinity peptide and an anti-IL-17A Fab as stabilizers, neither the production yield nor the characterization of the IL-17A/Fab complex was reported. To facilitate co-crystallization of IL-17A with small-molecule compounds derived from our DNA encoded library, we also describe the method for yield enhancement of anti-IL-17A Fab production and characterize the IL-17A/Fab complex for the first time, providing an essential prerequisite for structure-based drug discovery targeting IL-17A.

scholarly journals Alterations in Pattern Baldness According to Sex: Hair Metabolomics Approach

Metabolites ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 178
Author(s):  
Yu Ra Lee ◽  
Bark Lynn Lew ◽  
Woo Young Sim ◽  
Jongki Hong ◽  
Bong Chul Chung
Keyword(s):  
Metabolic Pathway ◽  
Metabolic Pathways ◽  
Ultra Performance Liquid Chromatography ◽  
Liquid Chromatography Mass Spectrometry ◽  
Hair Samples ◽  
Males And Females ◽  
And Control ◽  
First Time ◽  
Normal Controls ◽  
Male Hormone

Pattern baldness has been associated with the male hormone, dihydrotestosterone. In this study, we tried to determine how the overall metabolic pathways of pattern baldness differ in patients and in normal controls. Our study aimed to identify alterations in hair metabolomic profiles in order to identify possible markers of pattern baldness according to sex. Untargeted metabolomics profiling in pattern baldness patients and control subjects was conducted using ultra-performance liquid chromatography-mass spectrometry. To identify significantly altered metabolic pathways, partial least squares discriminant analysis was performed. Our analysis indicated differences in steroid biosynthesis pathway in both males and females. However, there was a remarkable difference in the androgen metabolic pathway in males, and the estrogen metabolic and arachidonic acid pathways in females. For the first time, we were able to confirm the metabolic pathway in pattern baldness patients using hair samples. Our finding improves understanding of pattern baldness and highlights the need to link pattern baldness and sex-related differences.

scholarly journals Synthesis and characterization of piperazine-substituted dihydrofuran derivatives viaMn(OAc)3 mediated radical cyclizations

2020 ◽  
Vol 44 (5) ◽  
pp. 1303-1313
Author(s):  
Sait SARI ◽  
Mehmet YILMAZ
Keyword(s):  
Synthesis And Characterization ◽  
Radical Cyclizations ◽  
Dicarbonyl Compounds ◽  
Piperazine Derivatives ◽  
High Yields ◽  
First Time

The aim of this study is to synthesize novel piperazine-containing dihydrofuran compounds (3a-n)from radical additions and cyclizations of diacyl and alkyl-acyl piperazine derivatives (1a-h) with 1,3-dicarbonyl compounds (2a-c) mediated by Mn(OAc)3 for the first time. From the reactions of 1a-c with dimedone (2a);1a, 1c, and 1d with acetylacetone (2b); and 1a with ethylacetoacetate(2c) ,the dihydrofuran-piperazine compounds 3a-c, 3d-f, and 3g were obtained in medium to high yields (31%–81%), respectively. In addition, dihydrofuran-piperazine compounds 3h-j and 3k-n were prepared at low to medium yields (20%–40%) from the reactions of 1e-g with 2a and 1e-h with 2c, respectively.

scholarly journals The Active Component of the Bioemulsifier Alasan from Acinetobacter radioresistens KA53 Is an OmpA-Like Protein

2002 ◽  
Vol 184 (1) ◽  
pp. 165-170 ◽  
Author(s):  
Amir Toren ◽  
Elisha Orr ◽  
Yossi Paitan ◽  
Eliora Z. Ron ◽  
Eugene Rosenberg
Keyword(s):  
Terminal Sequence ◽  
Emulsifying Activity ◽  
Active Protein ◽  
High Molecular Weight Complex ◽  
E Coli ◽  
Acinetobacter Radioresistens ◽  
Gene Coding ◽  
Surface Active ◽  
First Time ◽  
Solubilizing Activity

ABSTRACT The bioemulsifier of Acinetobacter radioresistens KA53, referred to as alasan, is a high-molecular-weight complex of polysaccharide and protein. Recently, one of the alasan proteins, with an apparent molecular mass of 45 kDa, was purified and shown to constitute most of the emulsifying activity. The N-terminal sequence of the 45-kDa protein showed high homology to an OmpA-like protein from Acinetobacter spp. In the research described here the gene coding for the 45-kDa protein was cloned, sequenced, and expressed in Escherichia coli. Recombinant protein AlnA (35.77 kDa without the leader sequence) had an amino acid sequence homologous to that of E. coli OmpA and contained 70% of the specific (hydrocarbon-in-water) emulsifying activity of the native 45-kDa protein and 2.4 times that of the alasan complex. In addition to their emulsifying activity, both the native 45-kDa protein and the recombinant AlnA were highly effective in solubilizing phenanthrene, ca. 80 μg per mg of protein, corresponding to 15 to 19 molecules of phenanthrene per molecule of protein. E. coli OmpA had no significant emulsifying or phenanthrene-solubilizing activity. The production of a recombinant surface-active protein (emulsification and solubilization of hydrocarbons in water) from a defined gene makes possible for the first time structure-function studies of a bioemulsan.

Monomer model: an integrated characterization method of geometrical deviations for assembly accuracy analysis

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Binbin Zhao ◽  
Yunlong Wang ◽  
Qingchao Sun ◽  
Yuanliang Zhang ◽  
Xiao Liang ◽  
...  
Keyword(s):  
Weak Links ◽  
Small Displacement ◽  
Accuracy Analysis ◽  
Content Type ◽  
Assembly Accuracy ◽  
Rotor Assembly ◽  
Relationship Of ◽  
Assembly Deviation ◽  
First Time

Purpose Assembly accuracy is the guarantee of mechanical product performance, and the characterization of the part with geometrical deviations is the basis of assembly accuracy analysis. Design/methodology/approach The existed small displacement torsors (SDT) model cannot fully describe the part with multiple mating surfaces, which increases the difficulty of accuracy analysis. This paper proposed an integrated characterization method for accuracy analysis. By analyzing the internal coupling relationship of the different geometrical deviations in a single part, the Monomer Model was established. Findings The effectiveness of the Monomer Model is verified through an analysis of a simulated rotor assembly analysis, and the corresponding accuracy analysis method based on the model reasonably predicts the assembly deviation of the rotor. Originality/value The Monomer Model realizes the reverse calculation of assembly deformation for the first time, which can be used to identify the weak links that affect the assembly accuracy, thus support the accuracy improvement in the re-assembly stage.

Skeletal elements of crinoid echinoderms: High-resolution structural and microanalytical results

1983 ◽  
Vol 41 ◽  
pp. 194-195
Author(s):  
D. F. Blake ◽  
L. F. Allard ◽  
D. R. Peacor
Keyword(s):  
High Resolution ◽  
Marine Invertebrates ◽  
Sea Urchins ◽  
Structural Features ◽  
Sea Stars ◽  
High Resolution Tem ◽  
Relationship Of ◽  
The Relationship ◽  
Insight Into

Echinodermata is a phylum of marine invertebrates which has been extant since Cambrian time (c.a. 500 m.y. before the present). Modern examples of echinoderms include sea urchins, sea stars, and sea lilies (crinoids). The endoskeletons of echinoderms are composed of plates or ossicles (Fig. 1) which are with few exceptions, porous, single crystals of high-magnesian calcite. Despite their single crystal nature, fracture surfaces do not exhibit the near-perfect {10.4} cleavage characteristic of inorganic calcite. This paradoxical mix of biogenic and inorganic features has prompted much recent work on echinoderm skeletal crystallography. Furthermore, fossil echinoderm hard parts comprise a volumetrically significant portion of some marine limestones sequences. The ultrastructural and microchemical characterization of modern skeletal material should lend insight into: 1). The nature of the biogenic processes involved, for example, the relationship of Mg heterogeneity to morphological and structural features in modern echinoderm material, and 2). The nature of the diagenetic changes undergone by their ancient, fossilized counterparts. In this study, high resolution TEM (HRTEM), high voltage TEM (HVTEM), and STEM microanalysis are used to characterize tha ultrastructural and microchemical composition of skeletal elements of the modern crinoid Neocrinus blakei.

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