Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii

Genomic sequence analysis of Acinetobacter baumannii revealed the presence of a putative Acid Phosphatase (AcpA; EC 3.1.3.2). A plasmid construct was made, and recombinant protein (rAcpA) was expressed in E. coli. PAGE analysis (carried out under denaturing/reducing conditions) of nickel-affinity purified protein revealed the presence of a near-homogeneous band of approximately 37 kDa. The identity of the 37 kDa species was verified as rAcpA by proteomic analysis with a molecular mass of 34.6 kDa from the deduced sequence. The dependence of substrate hydrolysis on pH was broad with an optimum observed at 6.0. Kinetic analysis revealed relatively high affinity for PNPP (Km = 90 μM) with Vmax, kcat, and Kcat/Km values of 19.2 pmoles s-1, 4.80 s-1(calculated on the basis of 37 kDa), and 5.30 x 104 M-1s-1, respectively. Sensitivity to a variety of reagents, i.e., detergents, reducing, and chelating agents as well as classic acid phosphatase inhibitors was examined in addition to assessment of hydrolysis of a number of phosphorylated compounds. Removal of phosphate from different phosphorylated compounds is supportive of broad, i.e., ‘nonspecific’ substrate specificity; although, the enzyme appears to prefer phosphotyrosine and/or peptides containing phosphotyrosine in comparison to serine and threonine. Examination of the primary sequence indicated the absence of signature sequences characteristic of Type A, B, and C nonspecific bacterial acid phosphatases.

Download Full-text

Intranasal Infection of Ferrets with SARS-CoV-2 as a Model for Asymptomatic Human Infection

Viruses ◽

10.3390/v13010113 ◽

2021 ◽

Vol 13 (1) ◽

pp. 113

Author(s):

Helen E. Everett ◽

Fabian Z. X. Lean ◽

Alexander M. P. Byrne ◽

Pauline M. van Diemen ◽

Shelley Rhodes ◽

...

Keyword(s):

Genomic Sequence ◽

Humoral Response ◽

Asymptomatic Infection ◽

Human Infection ◽

Viral Rna ◽

Post Inoculation ◽

Upper Respiratory Tract ◽

Infection Dynamics ◽

Genomic Sequence Analysis ◽

Upper Respiratory

Ferrets were experimentally inoculated with SARS-CoV-2 (severe acute respiratory syndrome (SARS)-related coronavirus 2) to assess infection dynamics and host response. During the resulting subclinical infection, viral RNA was monitored between 2 and 21 days post-inoculation (dpi), and reached a peak in the upper respiratory cavity between 4 and 6 dpi. Viral genomic sequence analysis in samples from three animals identified the Y453F nucleotide substitution relative to the inoculum. Viral RNA was also detected in environmental samples, specifically in swabs of ferret fur. Microscopy analysis revealed viral protein and RNA in upper respiratory tract tissues, notably in cells of the respiratory and olfactory mucosae of the nasal turbinates, including olfactory neuronal cells. Antibody responses to the spike and nucleoprotein were detected from 21 dpi, but virus-neutralizing activity was low. A second intranasal inoculation (re-exposure) of two ferrets after a 17-day interval did not produce re-initiation of viral RNA shedding, but did amplify the humoral response in one animal. Therefore, ferrets can be experimentally infected with SARS-CoV-2 to model human asymptomatic infection.

Download Full-text

Genomic Sequence Analysis of Granulovirus Isolated from the Tobacco Cutworm, Spodoptera litura

PLoS ONE ◽

10.1371/journal.pone.0028163 ◽

2011 ◽

Vol 6 (11) ◽

pp. e28163 ◽

Cited By ~ 15

Author(s):

Yong Wang ◽

Jae Young Choi ◽

Jong Yul Roh ◽

Qin Liu ◽

Xue Ying Tao ◽

...

Keyword(s):

Sequence Analysis ◽

Spodoptera Litura ◽

Genomic Sequence ◽

Tobacco Cutworm ◽

Genomic Sequence Analysis

Download Full-text

Identification of Purple Acid Phosphatase Inhibitors by Fragment-Based Screening: Promising New Leads for Osteoporosis Therapeutics

Chemical Biology & Drug Design ◽

10.1111/cbdd.12001 ◽

2012 ◽

Vol 80 (5) ◽

pp. 665-674 ◽

Cited By ~ 21

Author(s):

Daniel Feder ◽

Waleed M. Hussein ◽

Daniel J. Clayton ◽

Meng-Wei Kan ◽

Gerhard Schenk ◽

...

Keyword(s):

Acid Phosphatase ◽

Purple Acid Phosphatase ◽

Phosphatase Inhibitors

Download Full-text

Predicting Chromosome Flexibility from the Genomic Sequence Based on Deep Learning Neural Networks

Current Bioinformatics ◽

10.2174/1574893616666210827095829 ◽

2021 ◽

Vol 16 ◽

Author(s):

Jinghao Peng ◽

Jiajie Peng ◽

Haiyin Piao ◽

Zhang Luo ◽

Kelin Xia ◽

...

Keyword(s):

Deep Learning ◽

High Performance ◽

Genomic Sequence ◽

Sequence Data ◽

Function Analysis ◽

Double Helix ◽

Gm12878 Cell ◽

Genomic Sequence Analysis ◽

And Function ◽

Nuclear Processes

Background: The open and accessible regions of the chromosome are more likely to be bound by transcription factors which are important for nuclear processes and biological functions. Studying the change of chromosome flexibility can help to discover and analyze disease markers and improve the efficiency of clinical diagnosis. Current methods for predicting chromosome flexibility based on Hi-C data include the flexibility-rigidity index (FRI) and the Gaussian network model (GNM), which have been proposed to characterize chromosome flexibility. However, these methods require the chromosome structure data based on 3D biological experiments, which is time-consuming and expensive. Objective: Generally, the folding and curling of the double helix sequence of DNA have a great impact on chromosome flexibility and function. Motivated by the success of genomic sequence analysis in biomolecular function analysis, we hope to propose a method to predict chromosome flexibility only based on genomic sequence data. Method: We propose a new method (named "DeepCFP") using deep learning models to predict chromosome flexibility based on only genomic sequence features. The model has been tested in the GM12878 cell line. Results: The maximum accuracy of our model has reached 91%. The performance of DeepCFP is close to FRI and GNM. Conclusion: The DeepCFP can achieve high performance only based on genomic sequence.

Download Full-text

Phosphatase inhibitors—III. Benzylaminophosphonic acids as potent inhibitors of human prostatic acid phosphatase

Bioorganic & Medicinal Chemistry ◽

10.1016/0968-0896(96)00186-1 ◽

1996 ◽

Vol 4 (10) ◽

pp. 1693-1701 ◽

Cited By ~ 69

Author(s):

Scott A. Beers ◽

Charles F. Schwender ◽

Deborah A. Loughney ◽

Elizabeth Malloy ◽

Keith Demarest ◽

...

Keyword(s):

Acid Phosphatase ◽

Prostatic Acid Phosphatase ◽

Phosphatase Inhibitors

Download Full-text

N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Applied and Environmental Microbiology ◽

10.1128/aem.02881-15 ◽

2015 ◽

Vol 82 (4) ◽

pp. 1004-1014 ◽

Cited By ~ 23

Author(s):

Canfang Niu ◽

Huiying Luo ◽

Pengjun Shi ◽

Huoqing Huang ◽

Yaru Wang ◽

...

Keyword(s):

Acid Phosphatase ◽

Biochemical Characterization ◽

Animal Feed ◽

Glycosylation Site ◽

Site Directed Mutagenesis ◽

Acidic Ph ◽

Cleavage Sites ◽

Ph Optimum ◽

Content Type ◽

Histidine Acid Phosphatase

ABSTRACTN-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases fromYersinia kristensenii(YkAPPA) andYersinia rohdei(YrAPPA), each having anN-glycosylation motif, and one pepsin-sensitive HAP phytase fromYersinia enterocolitica(YeAPPA) that lacked anN-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering theN-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed inPichia pastorisfor biochemical characterization. Compared with those of theN-glycosylation site deletion mutants andN-deglycosylated enzymes, allN-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of theN-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of theN-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced inEscherichia colibut had no effect on the pepsin resistance ofN-glycosylated enzymes produced inP. pastoris. Thus,N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation ofN-glycosylation, for improvement of phytase properties for use in animal feed.

Download Full-text

Genomic sequence analysis of four new chrysanthemum virus B isolates: evidence of RNA recombination

Archives of Virology ◽

10.1007/s00705-011-1190-x ◽

2011 ◽

Vol 157 (3) ◽

pp. 531-537 ◽

Cited By ~ 14

Author(s):

Lakhmir Singh ◽

Vipin Hallan ◽

D. P. Martin ◽

Raja Ram ◽

A. A. Zaidi

Keyword(s):

Sequence Analysis ◽

Genomic Sequence ◽

Rna Recombination ◽

Genomic Sequence Analysis

Download Full-text

Identification and genomic sequence analysis of a new Spodoptera exigua multiple nucleopolyhedrovirus, SeMNPV-QD, isolated from Qingdao, China

Journal of Invertebrate Pathology ◽

10.1016/j.jip.2018.11.006 ◽

2019 ◽

Vol 160 ◽

pp. 8-17

Author(s):

Yingjian Chen ◽

Benxiang Qi ◽

Guiling Zheng ◽

Yuan Zhang ◽

Fei Deng ◽

...

Keyword(s):

Sequence Analysis ◽

Spodoptera Exigua ◽

Genomic Sequence ◽

Multiple Nucleopolyhedrovirus ◽

Genomic Sequence Analysis

Download Full-text

Identification and biochemical characterization of a novel PP2C-like Ser/Thr phosphatase inE. coli

10.1101/303594 ◽

2018 ◽

Author(s):

Krithika Rajagopalan ◽

Jonathan Dworkin

Keyword(s):

Biochemical Characterization ◽

Regulatory Protein ◽

Biochemical Properties ◽

Bacterial Genomes ◽

Phosphatase Inhibitors ◽

E Coli ◽

Specificity And Sensitivity ◽

Genes Encoding ◽

Number Of Bacteria

AbstractIn bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such asE. coli,which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Ser/Thr phosphatases belonging to several conserved families are found in bacteria. One family of particular interest are Ser/Thr phosphatases which have extensive sequence and structural homology to eukaryotic Ser/Thr PP2C phosphatases. These proteins, called eSTPs (eukaryotic-like Ser/Thr phosphatases), have been identified in a number of bacteria, but not inE. coli.Here, we describe a previously unknown eSTP encoded by anE. coliORF,yegK,and characterize its biochemical properties including its kinetics, substrate specificity and sensitivity to known phosphatase inhibitors. We investigate differences in the activity of this protein in closely relatedE. colistrains. Finally, we demonstrate that this eSTP acts to dephosphorylate a novel Ser/Thr kinase which is encoded in the same operon.ImportanceRegulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria including the model Gram-negative bacteriumE. colidemonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported inE. coli, no partner Ser/Thrphosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

Download Full-text

Analisis Bioinformatika Berbasis WEB untuk Eksplorasi Enzim Kitosanase Berdasarkan Kemiripan Sekuens

JURNAL Al-AZHAR INDONESIA SERI SAINS DAN TEKNOLOGI ◽

10.36722/sst.v1i4.84 ◽

2014 ◽

Vol 1 (4) ◽

pp. 197

Author(s):

Vanny Narita ◽

Arif Lelono Arum ◽

Siti Isnaeni M ◽

Nuri Y. Fawzya

Keyword(s):

Acinetobacter Baumannii ◽

Ribosomal Rna ◽

Staphylococcus Epidermidis ◽

Genomic Sequence ◽

Preliminary Test ◽

Gene Bank ◽

16S Ribosomal Rna ◽

Rna Sequences ◽

Web Based ◽

Aeromonas Veronii

Eksplorasi enzim secara tradisional dengan kultivasi mikroba sekarang ini tidak lagi efisien, karena menghabiskan waktu dan biaya. Bioinformatik berbasis web hadir untuk melakukan serangkaian analisis sekuen, baik itu DNA maupun protein, yang dapat digunakan sebagai penelitian pendahuluan, sehingga ekplorasi enzim menjadi lebih tepat sasaran. Penelitian ini telah melakukan analisis potongan sekuen 16S ribosomal RNA yang didapat dari 6 bakteri yang berasosiasi dengan udang. Analisis yang dilakukan adalah untuk mencari tahu tersedianya sekuen tersebut telah ada di Gene Bank atau merupakan strain baru khas Indonesia yang belum terpublikasi. Dengan menggunakan database 16S Microbial dan Reference Genomic Sequence, serta fasilitas BLAST nucleotide dan CLUSTALW2 didapatkan 5 nama bakteri yaitu Micromonospora sp. L5, Aeromonas veronii B565, Staphylococcus epidermidis ATCC 12228, Burkholderia sp. JV3, dan Acinetobacter baumanniiAB307-0294. Kelima mikroba ini memiliki tidak mempunyai gen kitosanase tetapi penyandi kitinase. Ketidakhadiran gen kitosanase dalam genome mikroba menjadikan mikroba unik untuk diketahui sekuens gen kitosanasenya, yang juga berpeluang untuk dipublikasikan.AbstractEnzymes exploration which is traditionally conducted by microbial cultivation, is no longer efficient, for spending the time and cost. Web based bioinformatics presents to do a series of sequence analysis, for query both DNA and protein, which can be used as preliminary test in order to direct the research effectively. We have conducted an analysis of 16S ribosomal RNA sequences from 6 bacteria in association with shrimp. The goal is finding out the recording in Gene Banks, which if they have not recorded means they are Indonesian strain. Using the 16S Microbial and Reference Genomic Sequence databases, as well as BLAST nucleotide and CLUSTALW2, we obtained 5 names of bacteria, i.e., Micromonospora sp. L5, Aeromonas veronii B565, Staphylococcus epidermidis ATCC 12228, Burkholderia sp. JV3, and Acinetobacter baumannii AB307-0294. These microbes do not have the chitosanase gene but have chitinase gene. The absence of chitosanase gene is unique its sequence that also gives opportunity for publication.

Download Full-text