scholarly journals Maximal Efficiency of Coupling between ATP Hydrolysis and Translocation of Polypeptides Mediated by SecB Requires Two Protomers of SecA

2008 ◽  
Vol 191 (3) ◽  
pp. 978-984 ◽  
Author(s):  
Chunfeng Mao ◽  
Simon J. S. Hardy ◽  
Linda L. Randall
Keyword(s):  
Escherichia Coli ◽  
Ternary Complex ◽  
Atp Hydrolysis ◽  
Protein Export ◽  
Maximal Efficiency ◽  
Low Efficiency ◽  
Hydrolysis Of ◽  
Low Activity

ABSTRACT SecA is the ATPase that provides energy for translocation of precursor polypeptides through the SecYEG translocon in Escherichia coli during protein export. We showed previously that when SecA receives the precursor from SecB, the ternary complex is fully active only when two protomers of SecA are bound. Here we used variants of SecA and of SecB that populate complexes containing two protomers of SecA to different degrees to examine both the hydrolysis of ATP and the translocation of polypeptides. We conclude that the low activity of the complexes with only one protomer is the result of a low efficiency of coupling between ATP hydrolysis and translocation.

scholarly journals ATP-Dependent Interactions between Escherichia coli Min Proteins and the Phospholipid Membrane In Vitro

2003 ◽  
Vol 185 (3) ◽  
pp. 735-749 ◽  
Author(s):  
Laura L. Lackner ◽  
David M. Raskin ◽  
Piet A. J. de Boer
Keyword(s):  
Escherichia Coli ◽  
Protein Dynamics ◽  
Atp Hydrolysis ◽  
Phospholipid Vesicles ◽  
Membrane Complex ◽  
Min Proteins ◽  
Specificity Factor ◽  
The Mind ◽  
Hydrolysis Of

ABSTRACT Proper placement of the division apparatus in Escherichia coli requires pole-to-pole oscillation of the MinC division inhibitor. MinC dynamics involves a membrane association-dissociation cycle that is driven by the activities of the MinD ATPase and the MinE topological specificity factor, which themselves undergo coupled oscillatory localization cycles. To understand the biochemical mechanisms underlying Min protein dynamics, we studied the interactions of purified Min proteins with phospholipid vesicles and the role of ATP in these interactions. We show that (i) the ATP-bound form of MinD (MinD.ATP) readily associates with phospholipid vesicles in the presence of Mg2+, whereas the ADP-bound form (MinD.ADP) does not; (ii) MinD.ATP binds membrane in a self-enhancing fashion; (iii) both MinC and MinE can be recruited to MinD.ATP-decorated vesicles; (iv) MinE stimulates dissociation of MinD.ATP from the membrane in a process requiring hydrolysis of the nucleotide; and (v) MinE stimulates dissociation of MinC from MinD.ATP-membrane complexes, even when ATP hydrolysis is blocked. The results support and extend recent work by Z. Hu et al. (Z. Hu, E. P. Gogol, and J. Lutkenhaus, Proc. Natl. Acad. Sci. USA 99:6761-6766, 2002) and support models of protein oscillation wherein MinE induces Min protein dynamics by stimulating the conversion of the membrane-bound form of MinD (MinD.ATP) to the cytoplasmic form (MinD.ADP). The results also indicate that MinE-stimulated dissociation of MinC from the MinC-MinD.ATP-membrane complex can, and may, occur prior to hydrolysis of the nucleotide.

scholarly journals Negative cooperativity in the nitrogenase Fe protein electron delivery cycle

2016 ◽  
Vol 113 (40) ◽  
pp. E5783-E5791 ◽  
Author(s):  
Karamatullah Danyal ◽  
Sudipta Shaw ◽  
Taylor R. Page ◽  
Simon Duval ◽  
Masaki Horitani ◽  
...  
Keyword(s):  
Ternary Complex ◽  
Atp Hydrolysis ◽  
Negative Cooperativity ◽  
Protein Hydrolysis ◽  
Kinetic Measurements ◽  
Mofe Protein ◽  
Fe Protein ◽  
Normal Mode Calculations ◽  
Protein Components ◽  
Hydrolysis Of

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen (N2) to two ammonia (NH3) molecules through the participation of its two protein components, the MoFe and Fe proteins. Electron transfer (ET) from the Fe protein to the catalytic MoFe protein involves a series of synchronized events requiring the transient association of one Fe protein with each αβ half of the α2β2 MoFe protein. This process is referred to as the Fe protein cycle and includes binding of two ATP to an Fe protein, association of an Fe protein with the MoFe protein, ET from the Fe protein to the MoFe protein, hydrolysis of the two ATP to two ADP and two Pi for each ET, Pi release, and dissociation of oxidized Fe protein-(ADP)2 from the MoFe protein. Because the MoFe protein tetramer has two separate αβ active units, it participates in two distinct Fe protein cycles. Quantitative kinetic measurements of ET, ATP hydrolysis, and Pi release during the presteady-state phase of electron delivery demonstrate that the two halves of the ternary complex between the MoFe protein and two reduced Fe protein-(ATP)2 do not undergo the Fe protein cycle independently. Instead, the data are globally fit with a two-branch negative-cooperativity kinetic model in which ET in one-half of the complex partially suppresses this process in the other. A possible mechanism for communication between the two halves of the nitrogenase complex is suggested by normal-mode calculations showing correlated and anticorrelated motions between the two halves.

F0 Cysteine, bCys21, in the Escherichia coli ATP Synthase Is Involved in Regulation of Potassium Uptake and Molecular Hydrogen Production in Anaerobic Conditions

2002 ◽  
Vol 22 (3-4) ◽  
pp. 421-430 ◽  
Author(s):  
Nelli Mnatsakanyan ◽  
Karine Bagramyan ◽  
Anait Vassilian ◽  
Robert K. Nakamoto ◽  
Armen Trchounian
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Production ◽  
Atp Synthase ◽  
Molecular Hydrogen ◽  
Atp Hydrolysis ◽  
Membrane Vesicles ◽  
Anaerobic Conditions ◽  
Whole Cells ◽  
B Subunit ◽  
Hydrolysis Of

The single cysteine in the b subunit of the membranous F0 sector and the 19 cysteines in extramembranous F1 sector of the Escherichia coli ATP synthase were replaced by alanine. When cells were grown under anaerobic conditions on glucose, the kcat for ATP hydrolysis of membrane vesicles containing the bCys21Ala mutant enzyme, but not enzymes with other cysteine replacements, was lower, while ATP-driven H+ pumping was unchanged. However, the ATP-dependent increase in the number of accessible thiol groups in membrane vesicles was negated. Furthermore, K+ uptake and molecular hydrogen production by whole cells and protoplasts was greatly decreased. These results indicate a role for the F0 subunit bCys21 in the functionality of F0F1 and coupling to other membranous activities under fermentative conditions.

scholarly journals ESBLs and resistance to ceftazidime/avibactam and ceftolozane/tazobactam combinations in Escherichia coli and Pseudomonas aeruginosa

2019 ◽  
Vol 74 (7) ◽  
pp. 1934-1939 ◽  
Author(s):  
José-Manuel Ortiz de la Rosa ◽  
Patrice Nordmann ◽  
Laurent Poirel
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
The Other ◽  
E Coli ◽  
Catalytic Activities ◽  
Excellent Activity ◽  
Encoding Genes ◽  
Hydrolysis Of ◽  
Low Activity ◽  
Lactamase Inhibitor

Abstract Objectives To evaluate the efficacy of the recently launched β-lactam/β-lactamase inhibitor combinations ceftazidime/avibactam and ceftolozane/tazobactam against ESBL-producing Escherichia coli and Pseudomonas aeruginosa strains. Methods A series of ESBL-encoding genes (blaTEM, blaSHV, blaCTX-M, blaVEB, blaPER, blaGES and blaBEL) was cloned and expressed in E. coli or P. aeruginosa recipient strains. Cultures of E. coli TOP10 harbouring recombinant plasmids and therefore producing the different ESBLs tested were grown in order to perform measurements of catalytic activities, using benzylpenicillin, ceftazidime and ceftolozane as substrates. IC50s were additionally determined for clavulanic acid, tazobactam and avibactam. Results We showed here an overall better activity of ceftazidime/avibactam compared with ceftolozane/tazobactam toward ESBL-producing E. coli and P. aeruginosa. Several ESBLs of the GES, PER and BEL types conferred resistance to ceftolozane/tazobactam in E. coli and P. aeruginosa. For GES-6 and PER-1 producers, resistance to ceftolozane/tazobactam could be explained by a high hydrolysis of ceftolozane and a low activity of tazobactam as an inhibitor. On the other hand, PER-producing P. aeruginosa also exhibited resistance to ceftazidime/avibactam. Conclusions Altogether, the results show that the ESBL PER-1, which is widespread worldwide, may be a source of resistance to both ceftolozane/tazobactam and ceftazidime/avibactam. Excellent activity of ceftazidime/avibactam was highlighted for both ESBL-producing E. coli and ESBL-producing P. aeruginosa.

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