The type IV pilus assembly motor PilB is a robust hexameric ATPase with complex kinetics

2018 ◽  
Vol 475 (11) ◽  
pp. 1979-1993 ◽  
Author(s):  
Andreas Sukmana ◽  
Zhaomin Yang
Keyword(s):  
Atpase Activity ◽  
Enrichment Culture ◽  
Physiological State ◽  
Substrate Inhibition ◽  
Kinetic Studies ◽  
Enzyme Analysis ◽  
Type Iv Pilus ◽  
Type Iv ◽  
High Concentrations

The bacterial type IV pilus (T4P) is a versatile nanomachine that functions in pathogenesis, biofilm formation, motility, and horizontal gene transfer. T4P assembly is powered by the motor ATPase PilB which is proposed to hydrolyze ATP by a symmetrical rotary mechanism. This mechanism, which is deduced from the structure of PilB, is untested. Here, we report the first kinetic studies of the PilB ATPase, supporting co-ordination among the protomers of this hexameric enzyme. Analysis of the genome sequence of Chloracidobacterium thermophilum identified a pilB gene whose protein we then heterologously expressed. This PilB formed a hexamer in solution and exhibited highly robust ATPase activity. It displays complex steady-state kinetics with an incline followed by a decline over an ATP concentration range of physiological relevance. The incline is multiphasic and the decline signifies substrate inhibition. These observations suggest that variations in intracellular ATP concentrations may regulate T4P assembly and T4P-mediated functions in vivo in accordance with the physiological state of bacteria with unanticipated complexity. We also identified a mutant pilB gene in the genomic DNA of C. thermophilum from an enrichment culture. The mutant PilB variant, which is significantly less active, exhibited similar inhibition of its ATPase activity by high concentrations of ATP. Our findings here with the PilB ATPase from C. thermophilum provide the first line of biochemical evidence for the co-ordination among PilB protomers consistent with the symmetrical rotary model of catalysis based on structural studies.

scholarly journals The PilB-PilZ-FimX regulatory complex of the Type IV pilus from Xanthomonas citri

2021 ◽  
Vol 17 (8) ◽  
pp. e1009808
Author(s):  
Edgar E. Llontop ◽  
William Cenens ◽  
Denize C. Favaro ◽  
Germán G. Sgro ◽  
Roberto K. Salinas ◽  
...  
Keyword(s):  
Stable Complex ◽  
Type Iv Pilus ◽  
Xanthomonas Citri ◽  
Inner Membrane ◽  
Type Iv ◽  
Binary Complexes ◽  
Consistent Model ◽  
Wide Range ◽  
Regulatory Complex

Type IV pili (T4P) are thin and flexible filaments found on the surface of a wide range of Gram-negative bacteria that undergo cycles of extension and retraction and participate in a variety of important functions related to lifestyle, defense and pathogenesis. During pilus extensions, the PilB ATPase energizes the polymerization of pilin monomers from the inner membrane. In Xanthomonas citri, two cytosolic proteins, PilZ and the c-di-GMP receptor FimX, are involved in the regulation of T4P biogenesis through interactions with PilB. In vivo fluorescence microscopy studies show that PilB, PilZ and FimX all colocalize to the leading poles of X. citri cells during twitching motility and that this colocalization is dependent on the presence of all three proteins. We demonstrate that full-length PilB, PilZ and FimX can interact to form a stable complex as can PilB N-terminal, PilZ and FimX C-terminal fragments. We present the crystal structures of two binary complexes: i) that of the PilB N-terminal domain, encompassing sub-domains ND0 and ND1, bound to PilZ and ii) PilZ bound to the FimX EAL domain within a larger fragment containing both GGDEF and EAL domains. Evaluation of PilZ interactions with PilB and the FimX EAL domain in these and previously published structures, in conjunction with mutagenesis studies and functional assays, allow us to propose an internally consistent model for the PilB-PilZ-FimX complex and its interactions with the PilM-PilN complex in the context of the inner membrane platform of the X. citri Type IV pilus.

Mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus: purification, characterization, and kinetic studies

1991 ◽  
Vol 69 (7) ◽  
pp. 454-459 ◽  
Author(s):  
J. I. de Vicente ◽  
P. del Valle ◽  
F. Busto ◽  
D. de Arriaga ◽  
J. Soler
Keyword(s):  
Atpase Activity ◽  
Enzyme Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Kinetic Studies ◽  
Kinetic Properties ◽  
Phycomyces Blakesleeanus ◽  
Molecular Weights ◽  
Allosteric Effector ◽  
High Concentrations

Mitochondrial F1-ATPase was purified from the mycelium of Phycomyces blakesleeanus NRRL 1555(−) and its kinetic characteristics were studied. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of the enzyme reveals five bands (α, β, γ, δ, and ε) characteristic of the F1 portion with apparent molecular weights of 60 000, 53 000, 31 000, 25 000, and 21 000, respectively. The molecular weight of the native F1-ATPase from Phycomyces blakesleeanus was in agreement with the stoichiometry α3 β3 γ δ ε. The MgATP complex is the true substrate for ATPase activity which has a Km value of 0.15 mM. High concentrations of free ATP or free Mg2+ ions inhibit the ATPase activity. ADP appears to act as a negative allosteric effector with regard to MgATP hydrolysis, with the apparent Vmax remaining unchanged.Key words: mitochondrial F1-ATPase, Phycomyces blakesleeanus, enzyme purification, kinetic properties.

Stability of nucleoside triphosphate levels in the red cells of the snake

1997 ◽  
Vol 200 (7) ◽  
pp. 1125-1131
Author(s):  
R Ingermann ◽  
D Bencic ◽  
J Herman
Keyword(s):  
Atpase Activity ◽  
Red Cells ◽  
Metabolic Inhibitors ◽  
Cell Swelling ◽  
Nucleoside Triphosphate ◽  
High Ph ◽  
High Concentrations ◽  
The Stability

Nucleated red cells in the nonpregnant garter snake (Thamnophis elegans) contain relatively high concentrations of nucleoside triphosphate (NTP), largely in the form of ATP, which is found at concentrations of approximately 10 mmol l-1 relative to cell volume and 15 mmol l-1 relative to cell water. During pregnancy, levels of NTP in adult red cells rise by approximately 50 % concomitant with an increase in blood progesterone level. The stability of the NTP pool within these red cells was assessed by maintaining cells in vitro at 20 °C, without exogenous nutrients, and in the presence and absence of the metabolic inhibitors iodoacetate and/or cyanide. After 96 h, NTP levels in adult red cells not exposed to the inhibitors had decreased by only approximately 10 %, while in the presence of both inhibitors NTP levels had fallen by less than 50 %. Red cell NTP levels were not affected by acute exposure to high concentrations of progesterone either in vivo or in vitro. NTP levels were much more labile when the cells were maintained in vitro at either low or high pH. Maintenance of red cells at pH 6.0 for 24 h resulted in a decrease in NTP levels of approximately 50 % and at pH 10.0 the levels fell by approximately 80 %, while buffers containing only ATP caused no detectable degradation. Incubation at low or high pH promoted some cell swelling; however, the magnitude of the decreases in intracellular NTP concentration prompted by these pH levels could not be mimicked by incubation of red cells in hypotonic buffer. Total nonspecific ATPase activity at pH 6.0 was approximately 55 % greater than that at pH 7.4, while at pH 10.0 it was approximately 6 % of that at pH 7.4. The pH-dependent decrease in intracellular NTP levels cannot, therefore, be due to stimulation of ATPase activity, at least not at high pH. Overall, the data are consistent with the hypothesis that an appreciable portion of the NTP within these cells is compartmentalized in a stable, but pH-sensitive, pool sequestered from intracellular ATP-hydrolyzing processes.

scholarly journals EULAR Scleroderma Trials and Research group statement and recommendations on endothelial precursor cells

2008 ◽  
Vol 68 (2) ◽  
pp. 163-168 ◽  
Author(s):  
J H W Distler ◽  
Y Allanore ◽  
J Avouac ◽  
R Giacomelli ◽  
S Guiducci ◽  
...  
Keyword(s):  
Expert Panel ◽  
Enrichment Culture ◽  
Precursor Cells ◽  
Future Research ◽  
Face To Face ◽  
Type Iv ◽  
Consensus Statements ◽  
Factor Type

Systemic sclerosis (SSc) is characterised by a progressive microangiopathy that contributes significantly to the morbidity of patients with SSc. Besides insufficient angiogenesis, defective vasculogenesis with altered numbers of endothelial precursor cells (EPCs) might also contribute to the vascular pathogenesis of SSc. However, different protocols for isolation, enrichment, culture and quantification of EPCs are currently used, which complicate comparison and interpretation of the results from different studies.The aim of the European League Against Rheumatism Scleroderma Trials and Research (EUSTAR) group expert panel was to provide recommendations for standardisation of future research on EPCs. Consensus statements and recommendations were developed in a face to face meeting by an expert panel of the basic science working group of EUSTAR.The findings were: cardiovascular risk factors and medications such as statins should be described in detail. A detailed description of methods considering isolation, culture, enrichment and detection of EPCs should be given. For in vitro culture of EPCs, no protocol has been shown to be superior to another, but coating with laminin and type IV collagen would resemble most closely the situation in vivo. The endothelial phenotype should be confirmed in all in vitro cultures at the end of the culture period. We recommend using CD133, vascular endothelial growth factor type 2 receptor (VEGFR2) and CD34 in combination with a viability marker for quantification of EPCs in the blood. Finally, exact standard operating procedures for fluorescence-activated cell sorting (FACS) analysis are given that should be strictly followed.In summary, the EUSTAR recommendations will help to unify EPC research and allow better comparison between the results of different studies.

scholarly journals Partial purification and regulatory properties of phosphofructokinase from Aspergillus niger

1983 ◽  
Vol 209 (3) ◽  
pp. 669-676 ◽  
Author(s):  
A Habison ◽  
C P Kubicek ◽  
M Röhr
Keyword(s):  
Citric Acid ◽  
Aspergillus Niger ◽  
Kinetic Studies ◽  
Maximal Activity ◽  
Distinct Species ◽  
Hill Coefficient ◽  
Partial Purification ◽  
Maximal Velocity ◽  
High Concentrations

Phosphofructokinase (EC 2.7.1.11) from a citric acid-producing strain of Aspergillus niger was partially purified by the application of affinity chromatography on Blue Dextran-Sepharose and the use of fructose 6-phosphate and glycerol as stabilizers in the working buffer. The resulting preparation was still impure, but free of enzyme activities interfering with kinetic investigations. Kinetic studies showed that the enzyme exhibits high co-operativity with fructose 6-phosphate, but shows Michaelis-Menten kinetics with ATP, which inhibits at concentrations higher than those for maximal activity. Citrate and phosphoenolpyruvate inhibit the enzyme; citrate increases the substrate (fructose 6-phosphate) concentration for half-maximal velocity, [S]0.5, and the Hill coefficient, h. The inhibition by citrate is counteracted by NH4+, AMP and phosphate. Among univalent cations tested only NH4+ activates by decreasing the [S]0.5 for fructose 6-phosphate and h, but has no effect on Vmax. AMP and ADP activate at low and inhibit at high concentrations of fructose 6-phosphate, thereby decreasing the [S]0.5 for fructose 6-phosphate. Phosphate has no effect in the absence of citrate. The results indicate that phosphofructokinase from A. niger is a distinct species of this enzyme, with some properties similar to those of the yeast enzyme and in some other properties resembling the mammalian enzyme. The results of determinations of activity at substrate and effector concentrations resembling the conditions that occur in vivo support the hypothesis that the apparent insensitivity of the enzyme to citrate during the accumulation of citric acid in the fungus is due to counteraction of citrate inhibition by NH4+.

scholarly journals Investigation of the in vitro and in vivo activity of the type IV pilus extension ATPase BfpD

2020 ◽  
Author(s):  
Jinlei Zhao ◽  
Shahista Nisa ◽  
Michael S. Donnenberg
Keyword(s):  
Atpase Activity ◽  
Loss Of Function ◽  
Wild Type ◽  
Multifunctional Protein ◽  
Mutant Proteins ◽  
E Coli ◽  
Type Iv ◽  
Kinetics Of

AbstractType IV pili (T4Ps) are multifunctional protein fibers found in many bacteria and archaea. All T4P systems have an extension ATPase, which provides the energy required to push structural subunits out of the membrane. We previously reported that the BfpD T4P ATPase from enteropathogenic E. coli (EPEC) has the expected hexameric structure and ATPase activity, the latter enhanced by the presence of the N-terminal cytoplasmic domains of its partner proteins BfpC and BfpE. In this study, we further investigated the kinetics of the BfpD ATPase. Despite high purity of the proteins, the reported enhanced ATPase activity was found to be from (an) ATPase(s) contaminating the N-BfpC preparation. Furthermore, although two mutations in highly conserved bfpD sites led to loss of function in vivo, the purified mutant proteins retained some ATPase activity, albeit less than the wild-type protein. Therefore, the observed ATPase activity of BfpD was also affected by (a) contaminating ATPase(s). Expression of the mutant bfpD alleles did not interfere with BfpD function in bacteria that also expressed wild-type BfpD. However, a similar mutation of bfpF, which encodes the retraction ATPase, blocked the function of wild-type BfpF when both were present. These results highlight similarities and differences in function and activity of T4P extension and retraction ATPases in EPEC.

The ATPase activity of an ‘essential’ Bacillus subtilis enzyme, YdiB, is required for its cellular function and is modulated by oligomerization

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 944-956 ◽  
Author(s):  
Johanna C. Karst ◽  
Anne-Emmanuelle Foucher ◽  
Tracey L. Campbell ◽  
Anne-Marie Di Guilmi ◽  
David Stroebel ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Atpase Activity ◽  
Normal Growth ◽  
Conditional Knockout ◽  
Protein Oligomerization ◽  
E Coli ◽  
Oligomeric Form ◽  
High Concentrations

Characterization of ‘unknown’ proteins is one of the challenges of the post-genomic era. Here, we report a study of Bacillus subtilis YdiB, which belongs to an uncharted class of bacterial P-loop ATPases. Precise deletion of the ydiB gene yielded a mutant with much reduced growth rate compared to the wild-type strain. In vitro, purified YdiB was in equilibrium among different forms, monomers, dimers and oligomers, and this equilibrium was strongly affected by salts; high concentrations of NaCl favoured the monomeric over the oligomeric form of the enzyme. Interestingly, the ATPase activity of the monomer was about three times higher than that of the oligomer, and the monomer showed a K m of about 60 μM for ATP and a V max of about 10 nmol min−1 (mg protein)−1 (k cat ∼10 h−1). This low ATPase activity was shown to be specific to YdiB because mutation of an invariant lysine residue in the P-loop motif (K41A) strongly attenuated this rate. This mutant was unable to restore a normal growth phenotype when introduced into a conditional knockout strain for ydiB, showing that the ATPase activity of YdiB is required for the in vivo function of the protein. Oligomerization was also observed with the purified YjeE from Escherichia coli, a YdiB orthologue, suggesting that this property is shared by all members of this family of ATPases. Importantly, dimers of YdiB were also observed in a B. subtilis extract, or when stabilized by formaldehyde cross-linking for YjeE from E. coli, suggesting that oligomerization might regulate the function of this new class of proteins in vivo.

scholarly journals PilB and PilT Are ATPases Acting Antagonistically in Type IV Pilus Function in Myxococcus xanthus

2008 ◽  
Vol 190 (7) ◽  
pp. 2411-2421 ◽  
Author(s):  
Vladimir Jakovljevic ◽  
Simone Leonardy ◽  
Michael Hoppert ◽  
Lotte Søgaard-Andersen
Keyword(s):  
Atpase Activity ◽  
Myxococcus Xanthus ◽  
Type Iv Pili ◽  
Atp Binding ◽  
Dynamic Surface ◽  
Conserved Residues ◽  
Type Iv ◽  
Atp Binding And Hydrolysis

ABSTRACT Type IV pili (T4P) are dynamic surface structures that undergo cycles of extension and retraction. T4P dynamics center on the PilB and PilT proteins, which are members of the secretion ATPase superfamily of proteins. Here, we show that PilB and PilT of the T4P system in Myxococcus xanthus have ATPase activity in vitro. Using a structure-guided approach, we systematically mutagenized PilB and PilT to resolve whether both ATP binding and hydrolysis are important for PilB and PilT function in vivo. PilB as well as PilT ATPase activity was abolished in vitro by replacement of conserved residues in the Walker A and Walker B boxes that are involved in ATP binding and hydrolysis, respectively. PilB proteins containing mutant Walker A or Walker B boxes were nonfunctional in vivo and unable to support T4P extension. PilT proteins containing mutant Walker A or Walker B boxes were also nonfunctional in vivo and unable to support T4P retraction. These data provide genetic evidence that both ATP binding and hydrolysis by PilB are essential for T4P extension and that both ATP binding and hydrolysis by PilT are essential for T4P retraction. Thus, PilB and PilT are ATPases that act at distinct steps in the T4P extension/retraction cycle in vivo.

Effects of 3-Amino-1,2,4-Triazole (AT) on Catalase and Other Compounds

1956 ◽  
Vol 186 (1) ◽  
pp. 19-23 ◽  
Author(s):  
Werner G. Heim ◽  
David Appleman ◽  
H. T. Pyfrom
Keyword(s):  
Catalase Activity ◽  
Physiological State ◽  
Relative Effect ◽  
Single Intraperitoneal Injection ◽  
Tissue Homogenates ◽  
Liver And Kidney ◽  
Liver Homogenates ◽  
High Concentrations

Rat liver and kidney, but not blood, catalase activity decreases profoundly within the first 3 hours after the intraperitoneal or intravenous injection of AT. AT administered orally to mice causes a reduction of liver catalase activity. The liver and kidney catalase activity of rats returns to normal about 7 days after a single intraperitoneal injection. Liver cytochrome c content, hemoglobin level and urinary urobilinogen excretion are not affected by AT administration. Liver peroxidase activity is decreased slightly 3 hours after injection of AT but returns to normal within 24 hours. Prolonged AT administration has no effect on the growth rate of young rats. AT reduces the catalase activity of plant tissue homogenates, liver homogenates and crystalline catalase in vitro but only at high concentrations. AT causes a reduction of chlorophyll content and catalase activity of plants when administered in vivo but the relative effect against these two constituents varies with species, physiological state and concentration.

Gastric H(+)-K(+)-ATPase activity is inhibited by reduction of disulfide bonds in beta-subunit

1992 ◽  
Vol 263 (1) ◽  
pp. C39-C46 ◽  
Author(s):  
D. C. Chow ◽  
C. M. Browning ◽  
J. G. Forte
Keyword(s):  
Atpase Activity ◽  
Disulfide Bonds ◽  
Elevated Temperatures ◽  
Sequence Data ◽  
Kinetic Studies ◽  
Monovalent Cation ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Cysteine Residues ◽  
High Concentrations

H(+)-K(+)-ATPase activity of rabbit isolated gastric microsomes was irreversibly inactivated by reducing agents, such as 2-mercaptoethanol and dithiothreitol. Similar to what has been observed for Na(+)-K(+)-ATPase, high concentrations of reagents, at moderately elevated temperatures, were required to inactivate H(+)-K(+)-ATPase, suggesting relative inaccessibility of the responsible disulfide bonds. Resistance against inactivation was conferred by monovalent cation activators of K(+)-stimulated ATPase and p-nitro-phenylphosphatase. The effectiveness of K+ congeners in protecting the enzyme was similar in sequence (Tl+ greater than K+ greater than Rb+) and concentration to their respective affinities for stimulating enzymatic activity, suggesting that the K(+)-bound form of the enzyme is more resistant to reduction than the free enzyme. Furthermore, Na+ antagonized the protective effect of K+. Labeling studies using fluorescein-maleimide indicated that 60-70% of the cysteine residues in the beta-subunit are in the oxidized form. Coupled with primary sequence data, this suggests that three disulfide bonds are present in the native beta-subunit. In contrast, less than 10% of the cysteine residues in the alpha-subunit are in the oxidized form. Kinetic studies showed that the 2-mercaptoethanol-induced loss of H(+)-K(+)-ATPase activity was correlated with a reduction of disulfide groups in the beta-subunit, while there was no significant change in the alpha-subunit. We conclude that reduction of disulfide bonds irreversibly inhibits H(+)-K(+)-ATPase activity, binding of K+ to the enzyme confers a resistance to disulfide bond reduction, and the responsible disulfide bonds are present in the beta-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

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