Functional Characterization of a Novel Member of the Amidohydrolase 2 Protein Family, 2-Hydroxy-1-Naphthoic Acid Nonoxidative Decarboxylase from Burkholderia sp. Strain BC1

ABSTRACTThe gene encoding a nonoxidative decarboxylase capable of catalyzing the transformation of 2-hydroxy-1-naphthoic acid (2H1NA) to 2-naphthol was identified, recombinantly expressed, and purified to homogeneity. The putative gene sequence of the decarboxylase (hndA) encodes a 316-amino-acid protein (HndA) with a predicted molecular mass of 34 kDa. HndA exhibited high identity with uncharacterized amidohydrolase 2 proteins of variousBurkholderiaspecies, whereas it showed a modest 27% identity with γ-resorcylate decarboxylase, a well-characterized nonoxidative decarboxylase belonging to the amidohydrolase superfamily. Biochemically characterized HndA demonstrated strict substrate specificity toward 2H1NA, whereas inhibition studies with HndA indicated the presence of zinc as the transition metal center, as confirmed by atomic absorption spectroscopy. A three-dimensional structural model of HndA, followed by docking analysis, identified the conserved metal-coordinating and substrate-binding residues, while their importance in catalysis was validated by site-directed mutagenesis.IMPORTANCEMicrobial nonoxidative decarboxylases play a crucial role in the metabolism of a large array of carboxy aromatic chemicals released into the environment from a variety of natural and anthropogenic sources. Among these, hydroxynaphthoic acids are usually encountered as pathway intermediates in the bacterial degradation of polycyclic aromatic hydrocarbons. The present study reveals biochemical and molecular characterization of a 2-hydroxy-1-naphthoic acid nonoxidative decarboxylase involved in an alternative metabolic pathway which can be classified as a member of the small repertoire of nonoxidative decarboxylases belonging to the amidohydrolase 2 family of proteins. The strict substrate specificity and sequence uniqueness make it a novel member of the metallo-dependent hydrolase superfamily.

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Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

Microorganisms ◽

10.3390/microorganisms9051107 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1107

Author(s):

Wonho Choi ◽

Yoshihiro Yamaguchi ◽

Ji-Young Park ◽

Sang-Hyun Park ◽

Hyeok-Won Lee ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Physiological Function ◽

Functional Characterization ◽

Site Directed Mutagenesis ◽

In Vitro Assays ◽

Cell Growth Inhibition ◽

The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

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Characterization of Gammaherpesvirus 68 Gene 50 Transcription

Journal of Virology ◽

10.1128/jvi.74.4.2029-2037.2000 ◽

2000 ◽

Vol 74 (4) ◽

pp. 2029-2037 ◽

Cited By ~ 71

Author(s):

Shaofan Liu ◽

Iglika V. Pavlova ◽

Herbert W. Virgin ◽

Samuel H. Speck

Keyword(s):

Virus Replication ◽

Functional Characterization ◽

Immediate Early Gene ◽

Early Gene ◽

Immediate Early ◽

Gene Target ◽

Putative Gene ◽

Murine Gammaherpesvirus ◽

Gammaherpesvirus 68

ABSTRACT Gene 50 is the only immediate-early gene that appears to be conserved among the characterized gammaherpesviruses. It has recently been demonstrated for the human viruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) that ectopic expression of the gene 50-encoded product in some latently infected cell lines can lead to the induction of virus replication, indicating that gene 50 is likely to play a pivotal role in regulating gammaherpesvirus reactivation. Here we demonstrate that the murine gammaherpesvirus 68 (γHV68) gene 50 is an immediate-early gene and that transcription of γHV68 gene 50 leads to the production of both spliced and unspliced forms of the gene 50 transcript. Splicing of the transcript near the 5′ end serves to extend the gene 50 open reading frame, as has been observed for the gene 50 transcripts encoded by KSHV and herpesvirus saimiri (Whitehouse et al., J. Virol. 71:2550–2554, 1997; Lukac et al., Virology 252:304–312, 1998; Sun et al., Proc. Natl. Acad. Sci. USA 95:10866–10871, 1998). Reverse transcription-PCR analyses, coupled with S1 nuclease protection assays, provided evidence that gene 50 transcripts initiate at several sites within the region from bp 66468 to 66502 in the γHV68 genome. Functional characterization of the region upstream of the putative gene 50 transcription initiation site demonstrated orientation-dependent promoter activity and identified a 110-bp region (bp 66442 to 66552) encoding the putative gene 50 promoter. Finally, we demonstrate that the γHV68 gene 50 can transactivate the γHV68 gene 57 promoter, a known early gene target of the gene 50-encoded transactivator in other gammaherpesviruses. These studies show that the γHV68 gene 50 shares several important molecular similarities with the gene 50 homologs in other gammaherpesviruses and thus provides an impetus for future studies analyzing the role of the γHV68 gene 50-encoded protein in acute virus replication and reactivation from latency in vivo.

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Functional Characterization of a Novel Promoter Element Required for an Innate Immune Response in Drosophila

Molecular and Cellular Biology ◽

10.1128/mcb.23.22.8272-8281.2003 ◽

2003 ◽

Vol 23 (22) ◽

pp. 8272-8281 ◽

Cited By ~ 21

Author(s):

Hanna Uvell ◽

Ylva Engström

Keyword(s):

Gene Expression ◽

Antimicrobial Peptide ◽

Functional Characterization ◽

Binding Activity ◽

Innate Immune ◽

Site Directed Mutagenesis ◽

Promoter Element ◽

Peptide Gene ◽

Inducible Genes

ABSTRACT Innate immune reactions are crucial processes of metazoans to protect the organism against overgrowth of faster replicating microorganisms. Drosophila melanogaster is a precious model for genetic and molecular studies of the innate immune system. In response to infection, the concerted action of a battery of antimicrobial peptides ensures efficient killing of the microbes. The induced gene expression relies on translocation of the Drosophila Rel transcription factors Relish, Dif, and Dorsal to the nucleus where they bind to κB-like motifs in the promoters of the inducible genes. We have identified another putative promoter element, called region 1 (R1), in a number of antimicrobial peptide genes. Site-directed mutagenesis of the R1 site diminished Cecropin A1 (CecA1) expression in transgenic Drosophila larvae and flies. Infection of flies induced a nuclear R1-binding activity that was unrelated to the κB-binding activity in the same extracts. Although the R1 motif was required for Rel protein-mediated CecA1 expression in cotransfection experiments, our data argue against it being a direct target for the Drosophila Rel proteins. We propose that the R1 and κB motifs are targets for distinct regulatory complexes that act in concert to promote high levels of antimicrobial peptide gene expression in response to infection.

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Functional characterization of two anti-estradiol antibodies as deduced from modelling and site-directed mutagenesis experiments

Journal of Molecular Recognition ◽

10.1002/jmr.525 ◽

2001 ◽

Vol 14 (2) ◽

pp. 99-109 ◽

Cited By ~ 4

Author(s):

Florence Bettsworth ◽

C�line Monnet ◽

B�n�dicte Watelet ◽

Nicole Battail-Poirot ◽

Bernard Gilquin ◽

...

Keyword(s):

Functional Characterization ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis

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The rare sugar N-acetylated viosamine is a major component of Mimivirus fibers

Journal of Biological Chemistry ◽

10.1074/jbc.m117.783217 ◽

2017 ◽

Vol 292 (18) ◽

pp. 7385-7394 ◽

Cited By ~ 7

Author(s):

Francesco Piacente ◽

Cristina De Castro ◽

Sandra Jeudy ◽

Matteo Gaglianone ◽

Maria Elena Laugieri ◽

...

Keyword(s):

Structural Model ◽

Functional Characterization ◽

Virus Genome ◽

Amino Group ◽

Rare Sugar ◽

Functional Annotations ◽

Terminal Domain ◽

Icosahedral Capsid

The giant virus Mimivirus encodes an autonomous glycosylation system that is thought to be responsible for the formation of complex and unusual glycans composing the fibers surrounding its icosahedral capsid, including the dideoxyhexose viosamine. Previous studies have identified a gene cluster in the virus genome, encoding enzymes involved in nucleotide-sugar production and glycan formation, but the functional characterization of these enzymes and the full identification of the glycans found in viral fibers remain incomplete. Because viosamine is typically found in acylated forms, we suspected that one of the genes might encode an acyltransferase, providing directions to our functional annotations. Bioinformatic analyses indicated that the L142 protein contains an N-terminal acyltransferase domain and a predicted C-terminal glycosyltransferase. Sequence analysis of the structural model of the L142 N-terminal domain indicated significant homology with some characterized sugar acetyltransferases that modify the C-4 amino group in the bacillosamine or perosamine biosynthetic pathways. Using mass spectrometry and NMR analyses, we confirmed that the L142 N-terminal domain is a sugar acetyltransferase, catalyzing the transfer of an acetyl moiety from acetyl-CoA to the C-4 amino group of UDP-d-viosamine. The presence of acetylated viosamine in vivo has also been confirmed on the glycosylated viral fibers, using GC-MS and NMR. This study represents the first report of a virally encoded sugar acetyltransferase.

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Purification and Functional Characterization of the C-Terminal Domain of the β-Actin-Binding Protein AIM1 In Vitro

Molecules ◽

10.3390/molecules23123281 ◽

2018 ◽

Vol 23 (12) ◽

pp. 3281 ◽

Cited By ~ 1

Author(s):

Fang Wu ◽

Liangkai Cheng ◽

Qi Yu ◽

Lin Zhang ◽

Hong Li ◽

...

Keyword(s):

Prostate Cancer ◽

Structural Model ◽

Functional Characterization ◽

Actin Binding ◽

Migration And Invasion ◽

Binding Modes ◽

C Terminus ◽

Biology Research

The protein absent in melanoma 1 (AIM1) is a member of the βγ-crystal lens superfamily that is associated with the development of multiple cancers. The binding of AIM1 to β-actin affects the migration and invasion of prostate cancer epithelial cells. The C-terminus of AIM1 is required for the β-actin interaction. However, the characteristics of AIM1 in vitro and the interaction mode between AIM1 and β-actin remain unknown. We describe novel methods to prepare pure recombinant AIM1 and identify possible binding modes between AIM1 and β-actin; we also obtain the crystal of the first two βγ-crystallin domains of AIM1 (g1g2) for future structural biology research. We first express and purify AIM1 after cloning the sequence into a modified pET-28a_psp expression vector. Next, we define the minimum unit formed by the βγ-crystallin domain repeats that bound to β-actin and perform its physiological function. Finally, we made the structural model of the AIM1 g1g2 that can be used to guide future biomedical investigations and prostate cancer research.

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Genetic and Functional Characterization of the Escherichia coli BarA-UvrY Two-Component System: Point Mutations in the HAMP Linker of the BarA Sensor Give a Dominant-Negative Phenotype

Journal of Bacteriology ◽

10.1128/jb.187.21.7317-7324.2005 ◽

2005 ◽

Vol 187 (21) ◽

pp. 7317-7324 ◽

Cited By ~ 24

Author(s):

Henrik Tomenius ◽

Anna-Karin Pernestig ◽

Claudia F. Méndez-Catalá ◽

Dimitris Georgellis ◽

Staffan Normark ◽

...

Keyword(s):

Signal Transduction ◽

Functional Characterization ◽

Point Mutations ◽

Dominant Negative ◽

Site Directed Mutagenesis ◽

Two Component System ◽

Component System ◽

Two Component

ABSTRACT The BarA-UvrY two-component system family is strongly associated with virulence but is poorly understood at the molecular level. During our attempts to complement a barA deletion mutant, we consistently generated various mutated BarA proteins. We reasoned that characterization of the mutants would help us to better understand the signal transduction mechanism in tripartite sensors. This was aided by the demonstrated ability to activate the UvrY regulator with acetyl phosphate independently of the BarA sensor. Many of the mutated BarA proteins had poor complementation activity but could counteract the activity of the wild-type sensor in a dominant-negative fashion. These proteins carried point mutations in or near the recently identified HAMP linker, previously implicated in signal transduction between the periplasm and cytoplasm. This created sensor proteins with an impaired kinase activity and a net dephosphorylating activity. Using further site-directed mutagenesis of a HAMP linker-mutated protein, we could demonstrate that the phosphoaccepting aspartate 718 and histidine 861 are crucial for the dephosphorylating activity. Additional analysis of the HAMP linker-mutated BarA sensors demonstrated that a dephosphorylating activity can operate via phosphotransfer within a tripartite sensor dimer in vivo. This also means that a tripartite sensor can be arranged as a dimer even in the dephosphorylating mode.

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Functional characterization of FlgM in the regulation of flagellar synthesis and motility in Yersinia pseudotuberculosis

Microbiology ◽

10.1099/mic.0.026294-0 ◽

2009 ◽

Vol 155 (6) ◽

pp. 1890-1900 ◽

Cited By ~ 18

Author(s):

Lisha Ding ◽

Yao Wang ◽

Yangbo Hu ◽

Steve Atkinson ◽

Paul Williams ◽

...

Keyword(s):

Yersinia Pseudotuberculosis ◽

Functional Characterization ◽

Direct Interaction ◽

Site Directed Mutagenesis ◽

Wild Type ◽

C Terminus ◽

In Trans ◽

Differential Gene ◽

Flagellar Biogenesis

We describe here the functional characterization of the flgM gene in Yersinia pseudotuberculosis. Direct interaction of FlgM with the alternative sigma factor σ 28 (FliA) was first confirmed. A conserved region in the C-terminus of FlgM was found which included the σ 28 binding domain. By site-directed mutagenesis, bacterial two-hybrid analysis and Western blotting, the primary FlgM binding sites with σ 28 were shown to be Ile85, Ala86 and Leu89. A role for FlgM in swimming motility was demonstrated by inactivation of flgM and subsequent complementation in trans. Transcriptional fusion analyses showed differential gene expression of flhDC, fliA, flgM and fliC in the fliA and flgM mutants compared with the wild-type. flhDC expression was not influenced by σ 28 or FlgM while fliA expression was abolished in the fliA mutant and considerably reduced in the flgM mutant when compared to the wild-type, indicating that both FliA and FlgM can activate fliA transcription. Conversely, flgM transcription was higher in the fliA mutant when compared to the wild-type, suggesting that flgM transcription was repressed by σ 28. Interestingly, fliC expression was markedly increased in the flgM mutant, suggesting a negative regulatory role for FlgM in fliC expression. The transcription of other σ-dependent genes (cheW, flgD, flaA, csrA and fliZ) was also examined in fliA and flgM mutant backgrounds and this revealed that other σ-factors apart from σ 28 may be involved in flagellar biogenesis in Y. pseudotuberculosis. Taking together the motility phenotypes and effects of flgM mutation on the regulation of these key motility genes, we propose that the mechanisms regulating flagellar biogenesis in Y. pseudotuberculosis may differ from those described for other bacteria.

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