Differential ligand-selective control of opposing enzymatic activities within a bifunctional c-di-GMP enzyme

2021 ◽  
Vol 118 (36) ◽  
pp. e2100657118
Author(s):  
Dayna C. Patterson ◽  
Myrrh Perez Ruiz ◽  
Hyerin Yoon ◽  
Johnnie A. Walker ◽  
Jean-Paul Armache ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Biofilm Formation ◽  
Structural Model ◽  
Structural Information ◽  
Enzymatic Activities ◽  
Guanosine Monophosphate ◽  
Negative Stain ◽  
Single Polypeptide

Cyclic dimeric guanosine monophosphate (c-di-GMP) serves as a second messenger that modulates bacterial cellular processes, including biofilm formation. While proteins containing both c-di-GMP synthesizing (GGDEF) and c-di-GMP hydrolyzing (EAL) domains are widely predicted in bacterial genomes, it is poorly understood how domains with opposing enzymatic activity are regulated within a single polypeptide. Herein, we report the characterization of a globin-coupled sensor protein (GCS) from Paenibacillus dendritiformis (DcpG) with bifunctional c-di-GMP enzymatic activity. DcpG contains a regulatory sensor globin domain linked to diguanylate cyclase (GGDEF) and phosphodiesterase (EAL) domains that are differentially regulated by gas binding to the heme; GGDEF domain activity is activated by the Fe(II)-NO state of the globin domain, while EAL domain activity is activated by the Fe(II)-O2 state. The in vitro activity of DcpG is mimicked in vivo by the biofilm formation of P. dendritiformis in response to gaseous environment, with nitric oxide conditions leading to the greatest amount of biofilm formation. The ability of DcpG to differentially control GGDEF and EAL domain activity in response to ligand binding is likely due to the unusual properties of the globin domain, including rapid ligand dissociation rates and high midpoint potentials. Using structural information from small-angle X-ray scattering and negative stain electron microscopy studies, we developed a structural model of DcpG, providing information about the regulatory mechanism. These studies provide information about full-length GCS protein architecture and insight into the mechanism by which a single regulatory domain can selectively control output domains with opposing enzymatic activities.

scholarly journals Insights into the Mn2+ Binding Site in the Agmatinase-Like Protein (ALP): A Critical Enzyme for the Regulation of Agmatine Levels in Mammals

2020 ◽  
Vol 21 (11) ◽  
pp. 4132
Author(s):  
María-Belen Reyes ◽  
José Martínez-Oyanedel ◽  
Camila Navarrete ◽  
Erika Mardones ◽  
Ignacio Martínez ◽  
...  
Keyword(s):  
Binding Site ◽  
Structural Model ◽  
Structural Information ◽  
Triple Mutant ◽  
Catalytically Active ◽  
New Type ◽  
Similar Affinity ◽  
Mammalian Protein

Agmatine is a neurotransmitter with anticonvulsant, anti-neurotoxic and antidepressant-like effects, in addition it has hypoglycemic actions. Agmatine is converted to putrescine and urea by agmatinase (AGM) and by an agmatinase-like protein (ALP), a new type of enzyme which is present in human and rodent brain tissues. Recombinant rat brain ALP is the only mammalian protein that exhibits significant agmatinase activity in vitro and generates putrescine under in vivo conditions. ALP, despite differing in amino acid sequence from all members of the ureohydrolase family, is strictly dependent on Mn2+ for catalytic activity. However, the Mn2+ ligands have not yet been identified due to the lack of structural information coupled with the low sequence identity that ALPs display with known ureohydrolases. In this work, we generated a structural model of the Mn2+ binding site of the ALP and we propose new putative Mn2+ ligands. Then, we cloned and expressed a sequence of 210 amino acids, here called the “central-ALP”, which include the putative ligands of Mn2+. The results suggest that the central-ALP is catalytically active, as agmatinase, with an unaltered Km for agmatine and a decreased kcat. Similar to wild-type ALP, central-ALP is activated by Mn2+ with a similar affinity. Besides, a simple mutant D217A, a double mutant E288A/K290A, and a triple mutant N213A/Q215A/D217A of these putative Mn2+ ligands result on the loss of ALP agmatinase activity. Our results indicate that the central-ALP contains the active site for agmatine hydrolysis, as well as that the residues identified are relevant for the ALP catalysis.

scholarly journals Gm14230 controls Tbc1d24 cytoophidia and neuronal cellular juvenescence

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0248517
Author(s):  
Takao Morimune ◽  
Ayami Tano ◽  
Yuya Tanaka ◽  
Haruka Yukiue ◽  
Takefumi Yamamoto ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Neuronal Cells ◽  
Epileptic Encephalopathy ◽  
Enzymatic Activities ◽  
Inosine Monophosphate Dehydrogenase ◽  
Inosine Monophosphate ◽  
Reaction Field ◽  
A Cell

It is not fully understood how enzymes are regulated in the tiny reaction field of a cell. Several enzymatic proteins form cytoophidia, a cellular macrostructure to titrate enzymatic activities. Here, we show that the epileptic encephalopathy-associated protein Tbc1d24 forms cytoophidia in neuronal cells both in vitro and in vivo. The Tbc1d24 cytoophidia are distinct from previously reported cytoophidia consisting of inosine monophosphate dehydrogenase (Impdh) or cytidine-5’-triphosphate synthase (Ctps). Tbc1d24 cytoophidia is induced by loss of cellular juvenescence caused by depletion of Gm14230, a juvenility-associated lncRNA (JALNC) and zeocin treatment. Cytoophidia formation is associated with impaired enzymatic activity of Tbc1d24. Thus, our findings reveal the property of Tbc1d24 to form cytoophidia to maintain neuronal cellular juvenescence.

The Hierarchic Order of Intermediate Filament Structure and Assembly

1985 ◽  
Vol 43 ◽  
pp. 722-725
Author(s):  
U. Aebi ◽  
E.C. Glavaris ◽  
R. Eichner
Keyword(s):  
Tissue Specificity ◽  
Structural Model ◽  
Eukaryotic Cells ◽  
Cdna Sequencing ◽  
Filament Structure ◽  
Keratin Filaments ◽  
Isoelectric Points ◽  
Immunological Crossreactivity

Five different classes of intermediate-sized filaments (IFs) have been identified in differentiated eukaryotic cells: vimentin in mesenchymal cells, desmin in muscle cells, neurofilaments in nerve cells, glial filaments in glial cells and keratin filaments in epithelial cells. Despite their tissue specificity, all IFs share several common attributes, including immunological crossreactivity, similar morphology (e.g. about 10 nm diameter - hence ‘10-nm filaments’) and the ability to reassemble in vitro from denatured subunits into filaments virtually indistinguishable from those observed in vivo. Further more, despite their proteinchemical heterogeneity (their MWs range from 40 kDa to 200 kDa and their isoelectric points from about 5 to 8), protein and cDNA sequencing of several IF polypeptides (for refs, see 1,2) have provided the framework for a common structural model of all IF subunits.

Kinase Targets for Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

2019 ◽  
Vol 12 (1) ◽  
pp. 27-49 ◽  
Author(s):  
Shahinda S.R. Alsayed ◽  
Chau C. Beh ◽  
Neil R. Foster ◽  
Alan D. Payne ◽  
Yu Yu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Enzymatic Activity ◽  
Bacterial Pathogen ◽  
Building Blocks ◽  
Mycolic Acid ◽  
Adaptive Responses ◽  
Mycolic Acids ◽  
Hydroxylated Fatty Acids

Background:Mycolic acids (MAs) are the characteristic, integral building blocks for the mycomembrane belonging to the insidious bacterial pathogen Mycobacterium tuberculosis (M.tb). These C60-C90 long α-alkyl-β-hydroxylated fatty acids provide protection to the tubercle bacilli against the outside threats, thus allowing its survival, virulence and resistance to the current antibacterial agents. In the post-genomic era, progress has been made towards understanding the crucial enzymatic machineries involved in the biosynthesis of MAs in M.tb. However, gaps still remain in the exact role of the phosphorylation and dephosphorylation of regulatory mechanisms within these systems. To date, a total of 11 serine-threonine protein kinases (STPKs) are found in M.tb. Most enzymes implicated in the MAs synthesis were found to be phosphorylated in vitro and/or in vivo. For instance, phosphorylation of KasA, KasB, mtFabH, InhA, MabA, and FadD32 downregulated their enzymatic activity, while phosphorylation of VirS increased its enzymatic activity. These observations suggest that the kinases and phosphatases system could play a role in M.tb adaptive responses and survival mechanisms in the human host. As the mycobacterial STPKs do not share a high sequence homology to the human’s, there have been some early drug discovery efforts towards developing potent and selective inhibitors.Objective:Recent updates to the kinases and phosphatases involved in the regulation of MAs biosynthesis will be presented in this mini-review, including their known small molecule inhibitors.Conclusion:Mycobacterial kinases and phosphatases involved in the MAs regulation may serve as a useful avenue for antitubercular therapy.

scholarly journals Biofilm formation in the lung contributes to virulence and drug tolerance of Mycobacterium tuberculosis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Poushali Chakraborty ◽  
Sapna Bajeli ◽  
Deepak Kaushal ◽  
Bishan Dass Radotra ◽  
Ashwani Kumar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Immune System ◽  
Biofilm Formation ◽  
Antimycobacterial Activity ◽  
Drug Tolerance ◽  
Host Immune System ◽  
Tissue Sections ◽  
Slow Growing

AbstractTuberculosis is a chronic disease that displays several features commonly associated with biofilm-associated infections: immune system evasion, antibiotic treatment failures, and recurrence of infection. However, although Mycobacterium tuberculosis (Mtb) can form cellulose-containing biofilms in vitro, it remains unclear whether biofilms are formed during infection in vivo. Here, we demonstrate the formation of Mtb biofilms in animal models of infection and in patients, and that biofilm formation can contribute to drug tolerance. First, we show that cellulose is also a structural component of the extracellular matrix of in vitro biofilms of fast and slow-growing nontuberculous mycobacteria. Then, we use cellulose as a biomarker to detect Mtb biofilms in the lungs of experimentally infected mice and non-human primates, as well as in lung tissue sections obtained from patients with tuberculosis. Mtb strains defective in biofilm formation are attenuated for survival in mice, suggesting that biofilms protect bacilli from the host immune system. Furthermore, the administration of nebulized cellulase enhances the antimycobacterial activity of isoniazid and rifampicin in infected mice, supporting a role for biofilms in phenotypic drug tolerance. Our findings thus indicate that Mtb biofilms are relevant to human tuberculosis.

scholarly journals Eap1p, an Adhesin That Mediates Candida albicans Biofilm Formation In Vitro and In Vivo

2007 ◽  
Vol 6 (6) ◽  
pp. 931-939 ◽  
Author(s):  
Fang Li ◽  
Michael J. Svarovsky ◽  
Amy J. Karlsson ◽  
Joel P. Wagner ◽  
Karen Marchillo ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Cell Adhesion ◽  
Biofilm Formation ◽  
Fungal Infections ◽  
Gene Dosage ◽  
Venous Catheter ◽  
Flow Chamber ◽  
Dependent Manner

ABSTRACT Candida albicans is the leading cause of systemic fungal infections in immunocompromised humans. The ability to form biofilms on surfaces in the host or on implanted medical devices enhances C. albicans virulence, leading to antimicrobial resistance and providing a reservoir for infection. Biofilm formation is a complex multicellular process consisting of cell adhesion, cell growth, morphogenic switching between yeast form and filamentous states, and quorum sensing. Here we describe the role of the C. albicans EAP1 gene, which encodes a glycosylphosphatidylinositol-anchored, glucan-cross-linked cell wall protein, in adhesion and biofilm formation in vitro and in vivo. Deleting EAP1 reduced cell adhesion to polystyrene and epithelial cells in a gene dosage-dependent manner. Furthermore, EAP1 expression was required for C. albicans biofilm formation in an in vitro parallel plate flow chamber model and in an in vivo rat central venous catheter model. EAP1 expression was upregulated in biofilm-associated cells in vitro and in vivo. Our results illustrate an association between Eap1p-mediated adhesion and biofilm formation in vitro and in vivo.

scholarly journals Attenuation of Aeromonas hydrophila Infection in Carassius auratus by YtnP, a N-acyl Homoserine Lactonase from Bacillus licheniformis T-1

Antibiotics ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 631
Author(s):  
Mengfan Peng ◽  
Wentao Tong ◽  
Zhen Zhao ◽  
Ling Xiao ◽  
Zhaoyue Wang ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Bacillus Licheniformis ◽  
Aeromonas Hydrophila ◽  
Biofilm Formation ◽  
Carassius Auratus ◽  
Quorum Quenching ◽  
Inhibition Rate ◽  
Sequence Identity

In this experiment, the quorum quenching gene ytnP of Bacillus licheniformis T-1 was cloned and expressed, and the effect against infection of Aeromonas hydrophila ATCC 7966 was evaluated in vitro and vivo. The BLAST results revealed a 99% sequence identity between the ytnP gene of T-1 and its homolog in B.subtilis sub sp. BSP1, and the dendroGram showed that the similarity in the YtnP protein in T-1 was 100% in comparison with B.subtilis 3610, which was categorized as the Aidc cluster of the MBL family. The AHL lactonase activity of the purified YtnP was detected as 1.097 ± 0.7 U/mL with C6-HSL as the substrate. Otherwise, purified YtnP protein could significantly inhibit the biofilm formation of A.hydrophila ATCC 7966 with an inhibition rate of 68%. The MIC of thiamphenicol and doxycycline hydrochloride against A. hydrophila reduced from 4 μg/mL and 0.5 μg/mL to 1 μg/mL and 0.125 μg/mL, respectively, in the presence of YtnP. In addition, YtnP significantly inhibited the expression of five virulence factors hem, ahyB, ast, ep, aerA of A. hydrophila ATCC 7966 as well (p < 0.05). The results of inhibition on virulence showed a time-dependence tendency, while the strongest anti-virulence effects were within 4–24 h. In vivo, when the YtnP protein was co-injected intraperitoneally with A. hydrophila ATCC 7966, it attenuated the pathogenicity of A. hydrophila and the accumulated mortality was 27 ± 4.14% at 96 h, which was significantly lower than the average mortality of 78 ± 2.57% of the Carassius auratus injected with 108 CFU/mL of A. hydrophila ATCC 7966 only (p < 0.001). In conclusion, the AHL lactonase in B. licheniformis T-1 was proven to be YtnP protein and could be developed into an agent against infection of A. hydrophila in aquaculture.

A novel polymorphism of human gene has an amino acid substitution (V365M) that decreases enzymatic activity in vitro and in vivo

2004 ◽  
Vol 76 (6) ◽  
pp. 519-527 ◽  
Author(s):  
T FUKAMI ◽  
M NAKAJIMA ◽  
R YOSHIDA ◽  
Y TSUCHIYA ◽  
Y FUJIKI ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Amino Acid Substitution ◽  
Human Gene

scholarly journals Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion

2007 ◽  
Vol 6 (12) ◽  
pp. 2214-2221 ◽  
Author(s):  
Lois M. Douglas ◽  
Li Li ◽  
Yang Yang ◽  
A. M. Dranginis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biofilm Formation ◽  
In Vitro System ◽  
Glycosylphosphatidylinositol Anchor ◽  
Fungal Adhesion ◽  
Very High ◽  
Molecular Weight Form

ABSTRACT The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

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