Functional Complexes In E. Coli

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degradosome

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The degradosome is a large, multiprotein complex involved in RNA degradation. It consists of the RNA degradation enzymes RNase E and PNPase, as well as the ATP-dependent RNA helicase RhlB and the metabolic enzyme enolase [ Py94 , Carpousis94 , Py96 ]. Polyphosphate kinase and the chaperone protein DnaK are also associated with and may be components of the degradosome [ Blum97 , Miczak96 ]. A "minimal" degradosome composed of only RNase E, PNPase and RhlB degrades malEF REP RNA in an ATP-dependent manner in vitro, with activity equivalent to purified whole degradosomes. RNase E enzymatic function is dispensible for this test case, whereas PNPase must be catalytically active and incorporated into the degradosome for degradation to occur [ Coburn99 ]. Based on immunogold labeling studies, RhlB and RNase E are present in equimolar quantities in the degradosome, which is tethered to the cytoplasmic membrane via the amino-terminus of RNase E [ Liou01 ].

RNase E provides the organizational structure for the degradosome. Its carboxy-terminal half binds PNPase, RhlB and enolase, and the loss of this portion of the protein prevents degradation of a number of degradosome substrates, including the ptsG and mukB mRNAs and RNA I [ Kido96 , Vanzo98 , Morita04 ]. This scaffold region is flexible, with isolated segments of increased structure that may be involved in binding other degradosome constituents [ Callaghan04 ]. RNase E binding to partner proteins can be selectively disrupted. Loss of RhlB and enolase binding results in reduced degradosome activity. Conversely, disrupted PNPase binding yields increased activity. Strains any alteration in RNase E binding do not grow as well as wild type [ Leroy02 ]. The amino-terminal half of RNase E contains sequences involved in oligomerization [ Vanzo98 ].

In vitro purified degradosome generates 147-nucleotide RNase E cleavage intermediates from rpsT mRNA. Continuous cycles of polyadenylation and PNPase cleavage are necessary and sufficient to break down these intermediates, though RNase II can block this second degradation step [ Coburn98 ]. RNAs with 3' REP stabilizers or stem loops must be polyadenylated to allow breakdown by the degradosome [ Khemici04 , Blum99 ]. Poly(G) and poly(U) tails do not allow degradation, though addition of a stretch of mixed nucleotides copied from within a coding region has stimulated degradation of a test substrate [ Blum99 ].

The degradosome copurifies with fragments from its RNA substrates, including rRNA fragments derived from cleavage of 16S and 23S rRNA by RNase E, 5S rRNA and ssrA RNA [ Bessarab98 , LinChao99 ].

The DEAD-box helicases SrmB, RhlE and CsdA bind RNase E in vitro at a different site than RhlB. RhlE and CsdA can both replace RhlB in promoting PNPase activity in vitro [ Khemici04a ]. CsdA is induced by cold shock, and following a shift to 15 degrees C it copurifies with the degradosome [ PrudhommeG04 ].

At least two poly(A)-binding proteins interact with the degradosome. The cold-shock protein CspE inhibits internal cleavage and breakdown of polyadenylated RNA by RNase E and PNPase by blocking digestion through the poly(A) tail. S1, a component of the 30S ribosome, binds to RNase E and PNPase without apparent effect on their activities [ Feng01 ].

The global effects of mutations in degradeosome constituents on mRNA levels have been evaluated using microarrays [ Bernstein04 ]

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GroEL-GroES chaperonin complex

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The Escherichia coli chaperone protein GroEL (Hsp60) and its regulator GroES are necessary for the proper folding of certain proteins [ Kusukawa89 ], [ Horwich93 ]. The crystal structure of GroEL has been determined to a resolution of 2.8 angstroms. GroEL forms an 800 kDa cylinder from two back-to-back heptameric rings of 57 kDa subunits [ Braig94 ]. X-ray crystallography to a resolution of 3.0 angstroms has determined the structure of GroEL-GroES complexed with seven ADP molecules. Hydrophobic residues of GroEL lining the interior of the rings are responsible for protein binding. GroES forms a heptamer of 10 kDa subunits which bind to the cis ring of the GroEL complex, forming a lid on the chamber [ Xu97 , Donald05 ]. Binding of GroES creates a large dome-shaped cavity with a highly polar inner surface in which a non-native protein has the opportunity to fold into its native form [ Xu97 ].

There are 85 cytosolic proteins identified by LC-MS/MS after purification that absolutely require GroEL for proper folding, 13 of which are essential. These substrates were enriched for the (βα)8 triosephosphate isomerase (TIM) barrel domain. There are another approximately 165 that are only partially dependent upon GroEL for proper folding [ Kerner05 ]. Sequence analysis has been performed to determine possible substrate recognition sequences for GroEL binding [ Chaudhuri05 ].

GroEL and GroES are both heat inducible but are also expressed constitutively and are required for growth under all conditions tested [ Fayet89 ]. In groEL temperature-sensitive mutants, a defined group of cytoplasmic proteins--including citrate synthase, ketoglutarate dehydrogenase, and polynucleotide phosphorylase--were translated but failed to reach native form [ Horwich93 ]. In vivo studies were conducted and it was determined that overproduction of either GroEL and GroES or DnaK and DnaJ prevents aggregation of misfolded proteins. From these studies, it was proposed that GroEL and GroES proteins and the DnaK and DnaJ proteins have complementary functions in the folding and assembly of most proteins [ Gragerov92 ]. Cellular localization studies, [ Gaitanaris94 ], found that although GroEL does associate transiently with newly synthesized proteins, it is absent from the ribosomes. This suggests that DnaK and DnaJ play an early role in protein maturation, whereas GroEL acts at a later stage.

Overexpressed GroEL/GroES promotes the folding of enzyme variants carrying mutations generated in vitro suggesting that it helps alleviate the destabilising constraints of protein mutations [ Tokuriki09 ]

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dipeptide ABC transporter

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The DppABCDF dipeptide transport system is a member of the ATP-Binding Cassette (ABC) Superfamily of transporters [ Wu95 ]. Based on sequence similarity, DppA is the substrate-binding component, while DppB and DppC are the membrane components, and DppD and DppF are the ATP-binding components of the ABC transporter. DppABCDF is similar in sequence and subunit composition to the oligopeptide uptake system OppABCDF, suggesting similar subunit functions.

DppA's unbound structure has been resolved by x-ray crystallography to resolutions of 3.2 Å [ Dunten93 ] and 2.0 Å, and shows two domains connected by two 'hinge' segments [ Nickitenko95 ]. The structure of DppA has also been determined with bound glycyl-L-leucine has been determined to a resolution of 3.2 Å [ Dunten95 ]. The structure reveals that the binding site recognizes the peptide backbone allowing for accommodation of various side chains [ Dunten95 ]. There is also a requirement for an unsubstituted α-amino group for transport of a peptide [ Gilvarg65 ].

Loss of DppA or DppBCDF resulted in pro mutants being unable to utilize Pro-Gly as a proline source [ Olson91 ]. Pro-Gly transport was inhibited by His-Glu, suggesting His-Glu is an additional substrate for DppABCDF [ Olson91 ]. Mutations in dpp displayed resistance to the toxic dipeptide Lys-aminoxyAla, the loss of ability to utilize Leu-Trp as a source of its required amino acids [ Payne84 ], resistance to Gly-Val, Leu-Val, and Val-Leu, and reduced uptake of Gly-Gly [ De73 ]. Substrate specificity of DppA was studied in a filter binding assay in which column fractions were monitored for binding activity towards radioactively labeled dipeptides and tripeptides. DppA was observed to mediate the ATP driven uptake of dipeptides and, to a lesser extent, tripeptides from the periplasm [ Smith99 ]. When an outer membrane heme receptor is expressed in E. coli, the dipeptide ABC transporter is also capable of transporting heme and the heme precursor, δ aminolevulinic acid, from the periplasm into the cytoplasm [ Letoffe06 ]. Binding of heme to purified DppA has been demonstrated [ Letoffe06 ].

DppA accumulates to high levels when grown in minimal media, but levels of DppA are reduced when the medium is supplemented with casamino acids [ Olson91 ]. DppA levels were decreased after 4 hours exposure to zinc stress [ Easton06 ] and in response to glucose limitation [ Wick01 ]. When grown in rich medium, gcvB deletion mutants had high constitutive expression of dppA compared to the parent strain [ Urbanowski00 ]. dppA expression is also repressed by PhoB during phosphate limitation [ Smith92 ]

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SufBC2D Fe-S cluster scaffold complex

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The assembly of iron-sulfur clusters requires complex biosynthetic machinery. E. coli encodes two sets of proteins, the Isc and the Suf system, to achieve this task. The SufBC2D complex functions as the scaffold for de novo assembly of Fe-S clusters [ Chahal09 ]; it can perform assembly of a [4Fe-4S] cluster in vitro and transfer it to target proteins [ Wollers10 ].

The SufBCD complex can be primarily isolated in a 1:2:1 stoichiometry of the SufB:SufC:SufD subunits; this complex can not be assembled in vitro from isolated components. When purified anaerobically, the complex contains 1 eq of FADH2; the complex does not bind the oxidized form, FAD [ Wollers10 ]

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cytochrome bo terminal oxidase

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The E. coli K-12 genome contains gene clusters for 3 cytochrome oxidase enzymes - cytochrome bo oxidase (CyoABCD), cytochrome bd-I oxidase (CydAB) and cytochrome bd-II oxidase (AppCD). The three enzymes function as the major terminal oxidases in the aerobic respiratory chain of E. coli. Cytochrome bo oxidase genes (cyoABCD) are expressed when oxygen levels are high while cytochrome bd-I oxidase genes are expressed under oxygen limited conditions. Both enymes contribute to the generation of a proton motive force (PMF), cytochrome bo oxidase functions as a proton pump whilst cytochrome bd-I does not [ Puustinen91 ]. Cytochrome bd-II does not contribute to the generation of a PMF and may function to uncouple catabolism from ATP synthesis.
The cytochrome bo terminal oxidase catalyzes the two-electron oxidation of ubiquinol within the membrane and the four-electron reduction of molecular oxygen to water. In the cell the enzyme functions as a proton pump, with a net movement of 2H+/e- across the cytoplasmic membrane, thereby generating a proton-motive force [ Puustinen91 ]. Expression of the cyo operon is negatively regulated by Fnr and the ArcA/ArcB two component system under anaerobic conditions [ Iuchi90a , Cotter90 ]. Expression varies depending on the carbon source used for growth - being highest on non-fermentable carbon sources and lowest on glucose [ Cotter90 ]. Expression is induced by iron limitation [ Cotter92 ].
Cytochrome bo terminal oxidase consists of four subunits encoded by the cyoB, cyoA, cyoC and cyoD genes, all of which are necessary for a functional enzyme. Analyses of intermediates in the assembly of cytochrome bo oxidase indicate that assembly of the complex is an ordered process whereby subunit III and IV assemble first, followed by subunit I and finally subunit II [ Stenberg07 ]. The cytochrome bo complex is similar to the aa3-type family of cytochrome c oxidases [ Chepuri90 ].
A crystal structure of the entire cytochrome bo terminal oxidase complex has been determined at 3.5 Å resolution [ Abramson00 ]

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ribosome

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30S: 16S rRNA (1542 nt – 11 modifications), 21 r-proteins
50S: 23S (2904 nt – 24 modifications) and 5S (120 nt), as well as 34 r-proteins
Takes up 40 % of the cell’s energy turn over
It is central to survival and linked to energy
20,000 – 90,000 ribosomes per cell

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DnaJ/DnaK/GrpE

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The DnaK system of E. coli is a homolog of the eukaryotic Hsp70 chaperone system. This chaperone system assists in a number of cytoplasmic cellular processes including folding of nascent polypeptide chains, rescue of misfolded proteins, polypeptide chain translocation through membranes, assembly and disassembly of protein complexes, and control of folded regulatory proteins' biological activity. The chaperone action of DnaK is powered by ATP hydrolysis and is assisted by partner chaperones DnaJ and GrpE [ Bukau98 ]. An in vitro system containing DnaK, DnaJ and GrpE plus ATP is able to convert misfolded luciferase into an unfolded intermediate which spontaneously refolds into its native configuration after release from the chaperone [ Sharma10 ].

Studies using peptides have shown that DnaK binds with the highest affinity with short hydrophobic segments in extended conformation [ Zhu96 ]. Screening a library of known DnaK chaperone substrates led to the determination of a consensus motif consisting of a hydrophobic core of 4-5 residues flanked by basic residues with Leu the most prominent (90%) within the hydrophobic core [ Bukau98 ].

Tryptophan fluorescence spectophotometry [ Ha95 ] indicates that the ATPase cycle of DnaK can be described as an alternation between two states: the ATP-bound and ADP-bound states. It is in the ATP-bound state that DnaK association with its substrate occurs.

Data generated by simulations of Hsp70-mediated protein folding under permanent and transient heat shock are in agreement with in vitro wild-type E. coli chaperone experimental data at 25°C. They also reflected the dynamics of ADP-bound state to ATP-bound state in response to temperature variations [ Hu07a ].

The DnaJ family members are a heterogeneous group of multidomain proteins all sharing a conserved ~80 amino acid domain, the J domain, located frequently near the NH2 terminus, which is critical for DnaK ATPase stimulation [ Wall94 ]. GrpE has been shown to be essential for regulation of release of ADP and Pi from DnaK, providing further stimulation for the turnover of ATP [ Liberek91 ]. It has also been shown that GrpE acts as a thermosensor in that at high temperatures there is a decreased efficacy of GrpE in catalyzing ATP/ADP turnover [ Grimshaw03 ].

Overproduction of DnaK/DnaJ caused a reduction in oxidative carbonylation of proteins [ Fredriksso05 ].

IbpA/IbpB, ClpB, and the DnaK system form a functional triade of chaperones [ Mogk03 ].

Subunit composition of DnaJ/DnaK/GrpE = [DnaJ][DnaK][GrpE]

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MukBEF complex

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Two conformations of the MukBEF complex appear to exist; the half-saturated complex, with a stoichiometry of B2(E2F)1, is relatively stable and can bind DNA, while the fully saturated MukBEF complex, with a stoichiometry of B2(E2F)2, is short-lived, unable to bind DNA and able to form multimeric complexes [ Petrushenk06a ].

The MukEF complex appears to compete with DNA for binding to MukB. The fully assembled MukBEF complex is unable to bind DNA; addition of MukEF to DNA-bound MukB displaces MukB from the DNA [ Petrushenk06a ].

Electron microscopy studies indicated that the MukE and MukF subunits of the MukBEF complex associate with the terminal globular domains of the MukB homodimer. The MukBEF complex can also be observed to form multimeric complexes in a variety of conformations [ Matoba05 ].

It was reported that he MukBEF complex can be detected in vitro only under conditions of increased Ca2+ or Mg2+ concentration [ Yamazoe99 ]; however, [ Matoba05 ] was able to purify the complex in the absence of ions.

A report that the MukBEF complex was able to compact a DNA molecule in an ATP binding-dependent manner [ Case04 ] was later retracted [ Case05 ].

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cytochrome bd-II terminal oxidase

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Synonyms: CbdAB, AppCB
Summary:
The E.coli K-12 genome contains gene clusters for 3 cytochrome oxidase enzymes - cytochrome bo oxidase (CyoABCD), cytochrome bd-I oxidase (CydAB) and cytochrome bd-II oxidase (AppCD). The three enzymes function as the major terminal oxidases in the aerobic respiratory chain of E. coli. Cytochrome bo oxidase genes (cyoABCD) are expressed when oxygen levels are high while cytochrome bd-I oxidase genes (cydAB) are expressed under oxygen limited conditions and both enzymes contribute to the generation of a proton motive force (PMF). The physiological role of cytochrome bd-II is less certain - whilst it does function as a quinol:oxygen oxidoreductase [ Sturr96 , Bekker09 ], it does not appear to contribute to the generation of a PMF [ Bekker09 ] and may play a role in uncoupling catabolism from ATP synthesis.

The two subunits of cytochrome bd-II encoded by the appC and appB genes are structurally similar to the cytochrome bd-I terminal oxidase CydA and CydB subunits respectively [ Dassa91 ]. The appCB-appA operon is under the control of the transcriptional activator AppY . It is induced upon entry into the stationary phase, as well as starvation for carbon and phosphate [ Atlung97 ]. Cytochrome bd-II oxidase is active under glucose-limited aerobic conditions and expression of cytochrome bd-II oxidase as a sole terminal oxidase allows growth on a highly reduced substrate such as glycerol [ Bekker09 ]. Flux analysis with respect to glucose catabolism and respiration suggests that an E. coli strain expressing cytochrome bd-II oxidase as a sole terminal oxidase synthesizes ATP by substrate level phosphorylation only and consequently does not translocate protons [ Bekker09 ].

Front 10

trimethylamine N-oxide reductase I

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There are three to four forms of trimethylamine N-oxide (TMAO) reductase in E. coli, one is a constitutive form and the others inducible. TorA and TorC constitute the inducible TMAO reductase I which can accept electrons from various physiological donors via several carriers. Unlike other anaerobic respiratory systems, which are only expressed under anaerobic conditions, TMAO reductase is expressed in both anaerobic and aerobic [ Ansaldi07 ] conditions. The enzyme contains molybdenum, iron, zinc and acid-labile sulfur. Ubiquinones may substitute for menaquinones in TMAO respiration [ Yamamoto86 , Barrett85 , Silvestro88 , Silvestro89 ].

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degradosome

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The degradosome is a large, multiprotein complex involved in RNA degradation. It consists of the RNA degradation enzymes RNase E and PNPase, as well as the ATP-dependent RNA helicase RhlB and the metabolic enzyme enolase [ Py94 , Carpousis94 , Py96 ]. Polyphosphate kinase and the chaperone protein DnaK are also associated with and may be components of the degradosome [ Blum97 , Miczak96 ]. A "minimal" degradosome composed of only RNase E, PNPase and RhlB degrades malEF REP RNA in an ATP-dependent manner in vitro, with activity equivalent to purified whole degradosomes. RNase E enzymatic function is dispensible for this test case, whereas PNPase must be catalytically active and incorporated into the degradosome for degradation to occur [ Coburn99 ]. Based on immunogold labeling studies, RhlB and RNase E are present in equimolar quantities in the degradosome, which is tethered to the cytoplasmic membrane via the amino-terminus of RNase E [ Liou01 ].
RNase E provides the organizational structure for the degradosome. Its carboxy-terminal half binds PNPase, RhlB and enolase, and the loss of this portion of the protein prevents degradation of a number of degradosome substrates, including the ptsG and mukB mRNAs and RNA I [ Kido96 , Vanzo98 , Morita04 ]. This scaffold region is flexible, with isolated segments of increased structure that may be involved in binding other degradosome constituents [ Callaghan04 ]. RNase E binding to partner proteins can be selectively disrupted. Loss of RhlB and enolase binding results in reduced degradosome activity. Conversely, disrupted PNPase binding yields increased activity. Strains any alteration in RNase E binding do not grow as well as wild type [ Leroy02 ]. The amino-terminal half of RNase E contains sequences involved in oligomerization [ Vanzo98 ].
In vitro purified degradosome generates 147-nucleotide RNase E cleavage intermediates from rpsT mRNA. Continuous cycles of polyadenylation and PNPase cleavage are necessary and sufficient to break down these intermediates, though RNase II can block this second degradation step [ Coburn98 ]. RNAs with 3' REP stabilizers or stem loops must be polyadenylated to allow breakdown by the degradosome [ Khemici04 , Blum99 ]. Poly(G) and poly(U) tails do not allow degradation, though addition of a stretch of mixed nucleotides copied from within a coding region has stimulated degradation of a test substrate [ Blum99 ].
The degradosome copurifies with fragments from its RNA substrates, including rRNA fragments derived from cleavage of 16S and 23S rRNA by RNase E, 5S rRNA and ssrA RNA [ Bessarab98 , LinChao99 ].
The DEAD-box helicases SrmB, RhlE and CsdA bind RNase E in vitro at a different site than RhlB. RhlE and CsdA can both replace RhlB in promoting PNPase activity in vitro [ Khemici04a ]. CsdA is induced by cold shock, and following a shift to 15 degrees C it copurifies with the degradosome [ PrudhommeG04 ].
At least two poly(A)-binding proteins interact with the degradosome. The cold-shock protein CspE inhibits internal cleavage and breakdown of polyadenylated RNA by RNase E and PNPase by blocking digestion through the poly(A) tail. S1, a component of the 30S ribosome, binds to RNase E and PNPase without apparent effect on their activities [ Feng01 ].
The global effects of mutations in degradeosome constituents on mRNA levels have been evaluated using microarrays [ Bernstein04 ].

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longitudinal peptidoglycan synthesis/chromosome segregation-directing complex

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The mre genes are responsible for rod shape and mecillinam sensitivity in E. coli [ Wachi87 , Wachi89 ]. Immunofluorescence microscopy has shown that MreB, an actin homolog, forms helical filaments beneath the surface of the cell [ Kruse03 ]. Filament formation has been shown to be dependent upon the rod-shape of the cell [ Kruse05 ]. MreB is also incorporated into cytoskeletal rings that are located near the midcell during cell division [ Vats07 ].
MreB is responsible for proper chromosome segregation and movement. Overexpression of MreB inhibits cell division. Overexpression of dysfunctional MreB results in altered MreB filament morphology, inhibition of cell division, mislocalized origin and terminus regions of the chromosome, and perturbed DNA segregation [ Kruse03 ].
SetB is involved in protein segregation and likely acts somewhere in the linkage of chromosomes to the force required to separate them [ Espeli03 ]. Co-expression of SetB-GFP and Myc-MreB showed that the two proteins co-localized and yeast two-hybrid experiments revealed that SetB and MreB interact.
Overexpression of ftsQAZ suppresses the lethality of MreBCD depletion by increasing the supply of monomers for the enlarged Z ring of round cells during division. Fractionation and GFP fusions studies have shown MreC and MreD associate with the inner membrane. Two-hybrid experiments have shown that MreBCD form a complex in which MreB interacts with itself and MreC, and MreC interacts with itself and MreD.
The coiled-coil domain of MreC is believed to allow it to dimerize while its alpha helices are embedded within the inner membrane. MreD is predicted to be membrane bound with five transmembrane α-helices. The cytoplasmic 13 -14 N-terminal amino acids of MreC are believed to interact with MreB lying just beneath the cell surface. MreC is also believed to interact with PBP2, which is responsible for lateral wall peptidoglycan synthesis, suggesting a role for the MreBCD complex in directing peptidoglycan formation through this interaction [ Kruse05 ]. Immunofluorescence microscopy has shown PBP2 localization in the cell periphery in band-like structures is similar to MreB localization and is dependent upon MreB in Caulobacter crescentus [ Figge04 ]. and E.coli [ Vats09 ]. Immunofluorescence microscopy has also shown that assembly of MreB, MreC and MreD into the cytoskeletal rings and coiled structures occurs independently. [ Vats09 ].

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Sec Translocation Complex

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The Sec translocation complex of E.coli K-12 is a large multisubunit enzyme that mediates the transport of nascent polypeptides across, or their integration into, the cytoplasmic membrane. The translocase consists of the inner membrane heterotrimeric SecYEG complex that forms the protein conducting channel plus an ancillary complex, SecDFYajC and the Yid C membrane protein, both of which interact with SecYEG to enhance protein transport or integration. The energy for protein translocation is provided by the motor protein ATPase SecA and the proton motive force (PMF).

Two pathways of protein translocation converge at the Sec translocon. In the posttranslational pathway the newly synthesised polypeptide is bound by SecB, a cytosolic chaperone which aids targeting to the membrane and maintains a translocation competent conformation of the pre-protein, while in the co-translational pathway the SRP complex binds to the nascent protein as it emerges from the ribosome and the SRP/ribosome/protein complex is then targeted to the Sec translocase.

An experimental approach using alkaline phosphatase (PhoA) fusions to protein signal sequences has allowed discrimination between the major modes of transport, including the Sec protein translocase, across the inner membrane [ Marrichi08 ].

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TatABCE protein export complex

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The twin-arginine translocation (Tat) system works in parallel with the E. coli Sec translocation system to transport folded proteins across the cytoplasmic membrane [ Weiner98 ]. While the Sec system transports unfolded proteins, Tat translocase functions to move structured macromolecular substrates, usually containing cofactors, across the cytoplasmic membrane. These proteins include those critical for bacterial respiratory and photosynthetic energy metabolism. Substrates utilizing the Tat pathway are characterized by essentially invariant amino-terminal sequences which contain consecutive arginine residues.
Cofactor-containing Tat substrates acquire their cofactors in the cytoplasm where they attain a folded conformation [ Berks00 ]. In studies using mutated tat strains [ Sargent98a ], precursor proteins that accumulate in the cytoplasm contain cofactors. In even more direct studies using folded green fluorescent protein (GFP) fused with the Tat signal peptide [ Santini01 ], the GFP was found to localize to the periplasm.
The Tat apparatus in E. coli is encoded by genes located in two genetic loci. The tatA operon encodes tatABCD; tatE is coded for in a separate locus [ Bogsch98 ]. TatA, TatB and TatE are similar in structure, predicted to comprise a membrane-spanning alpha helix at the amino terminus, followed by an amphipathic helix at the cytoplasmic side of the membrane and a variable-length carboxy terminus. TatA has been shown to be a fully integral membrane protein [ De01 ]. TatA and TatE share 50% sequence identity and share overlapping functions in Tat translocation. Deletion of either of these genes results in a decrease in the range of substrates, while deletions in both results in complete loss of Tat-dependent export [ Sargent98a ]. Although TatB has 20% sequence identity with TatA/TatE, it serves a distinct function in export. A deletion mutation of tatB alone is enough to completely abolish the translocation of some but not all endogenous Tat substrates [ Ize02 ]. TatC has similarly been shown to be essential for Tat-dependent protein export [ Bogsch98 ]. tatD encodes a soluble cytoplasmic protein with nuclease activity [ Wexler00 ]. Deletion studies of tatD and two of its homologues indicate that TatD family proteins are not essential for Tat-dependent protein translocation.
Functionally, protein translocation in the Tat system is energized exclusively by a transmembrane proton electrochemical gradient with no involvement of nucleotide hydrolysis [ Mould91 ] unlike the Sec translocation system which is powered by ATP hydrolysis.
Localization studies using fusion proteins with green fluorescent protein (GFP) demonstrated that TatA, TatB and TatC proteins all localize to the cellular poles, suggesting that active translocon poles are primarily located at polar positions in E. coli [ Berthelman04 ].
Within the purified Tat complex, TatB and TatC are present in a strict 1:1 ration and a TatBC fusion protein supports Tat dependent transport [ Bolhuis01 ]. Three-dimensional structures of TatBC-substrate complexes and unliganded TatBC have been obtained by single particle electron microscopy. The structures show substrate binding on the periphery of the TatBC complex and suggest that TatBC undergoes structural reorganisation upon substrate binding [ Tarry09 ].
An experimental approach using alkaline phosphatase (PhoA) fusions to protein signal sequences has allowed discrimination between the major modes of transport, including the Tat protein export system, across the inner membrane [ Marrichi08 ].

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L-lactate dehydrogenase

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Synonyms: lactic acid dehydrogenase

an oxidized electron acceptor + (S)-lactate <=> a reduced electron acceptor + pyruvate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is favored in the direction shown.

Alternative Substrates for (S)-lactate: L-2-hydroxybutyrate [ Futai77 ]

In Pathways: superpathway of fucose and rhamnose degradation , methylglyoxal degradation IV , superpathway of methylglyoxal degradation , L-lactaldehyde degradation (aerobic)
Summary:
In the presence of various phospholipids, the Km of the enzyme for L-lactate increases, but the vmax increases as well [ Kimura78 ].
The physiological electron acceptor appears to be unknown. Enzyme assays were performed using the non-physiological electron acceptor MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) [ Futai77 , Kimura78 , Nishimura83 ].
Cofactors or Prosthetic Groups: FMN [Comment 2 , Futai77 ]
Activators (Unknown Mechanism): an L-1-phosphatidylglycerol-phosphate [ Kimura78 ]
Inhibitors (Unknown Mechanism): 2-hydroxy-3-butynoate [ Walsh72 , Helmward89 ]
KM for (S)-lactate: 120 μM [ Futai77 ]
pH(opt): 8-9 [Futai77 ]

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Enzymatic reaction of: ribosomal-protein-S18-alanine N-acetyltransferase

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Synonyms: ribosomal-protein-alanine N-acetyltransferase, ribosomal protein S18 acetyltransferase, acetyl-CoA:ribosomal-protein-L-alanine N-acetyltransferase

a ribosomal protein-L-alanine + acetyl-CoA <=> a ribosomal protein-N-acetyl-L-alanine + coenzyme A

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

Reversibility of this reaction is unspecified.

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murein tripeptide ABC transporter

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OppABCDF is an ATP-dependent oligopeptide transporter that is a member of the ATP-Binding Cassette (ABC) Superfamily of transporters [ Pearce92 ]. Based on sequence similarity, OppB and OppC are the membrane components of the ABC transporter, and OppD and OppF are the ATP-binding components of the ABC transporter [ Angelopoul96 , Pearce92 ]. OppA is the periplasmic substrate-binding component, however MppA can replace OppA as a periplasmic-binding component of the transporter when it binds murein tripeptides [ Park98d ].

Binding affinity and competition assays have shown that OppABCDF will transport oligopeptides up to five amino acids in length, but has no affinity for free amino acids [ Guyer86 , Angelopoul96 ]. The system has been observed to function in oligopeptide uptake, as well as recycling of cell wall peptides [ Goodell87 , Hiles87 ]. MppA was shown to be required for murein tripeptide transport in a diaminopimelic acid-requiring strain [ Park98d ]. OppD has been purified and identified by direct assays as an ATP-binding component of the OppABCDF oligopeptide transporter [ Higgins85 ]. Insertional mutants of each of the opp genes were constructed, and the opp-minus strains were unable to utilize the peptide Pro-Gly-Gly, normally transported by the wild-type transporter [ Hiles87 ].

When an outer membrane heme receptor is expressed in E. coli, the murein tripeptide ABC transporter is also capable of transporting heme and the heme precursor, δ aminolevulinic acid, from the periplasm into the cytoplasm [ Letoffe06 ]. Binding of heme to purified MppA has been demonstrated and is inhibited by Pro-Phe-Lys [ Letoffe06 ].

Expression of oppABCD increased after long-term adaptation to growth in complex medium with acetate or propionate [ Polen03 ]. Expression of mppA decreased after long-term adaptation to growth in complex medium with acetate or propionate [ Polen03 ]. Expression of mppA was shown to be activated by cyclic AMP receptor protein [ Zheng04 ].

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dipeptide ABC transporter

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The DppABCDF dipeptide transport system is a member of the ATP-Binding Cassette (ABC) Superfamily of transporters [ Wu95 ]. Based on sequence similarity, DppA is the substrate-binding component, while DppB and DppC are the membrane components, and DppD and DppF are the ATP-binding components of the ABC transporter. DppABCDF is similar in sequence and subunit composition to the oligopeptide uptake system OppABCDF, suggesting similar subunit functions.

DppA's unbound structure has been resolved by x-ray crystallography to resolutions of 3.2 Å [ Dunten93 ] and 2.0 Å, and shows two domains connected by two 'hinge' segments [ Nickitenko95 ]. The structure of DppA has also been determined with bound glycyl-L-leucine has been determined to a resolution of 3.2 Å [ Dunten95 ]. The structure reveals that the binding site recognizes the peptide backbone allowing for accommodation of various side chains [ Dunten95 ]. There is also a requirement for an unsubstituted α-amino group for transport of a peptide [ Gilvarg65 ].

Loss of DppA or DppBCDF resulted in pro mutants being unable to utilize Pro-Gly as a proline source [ Olson91 ]. Pro-Gly transport was inhibited by His-Glu, suggesting His-Glu is an additional substrate for DppABCDF [ Olson91 ]. Mutations in dpp displayed resistance to the toxic dipeptide Lys-aminoxyAla, the loss of ability to utilize Leu-Trp as a source of its required amino acids [ Payne84 ], resistance to Gly-Val, Leu-Val, and Val-Leu, and reduced uptake of Gly-Gly [ De73 ]. Substrate specificity of DppA was studied in a filter binding assay in which column fractions were monitored for binding activity towards radioactively labeled dipeptides and tripeptides. DppA was observed to mediate the ATP driven uptake of dipeptides and, to a lesser extent, tripeptides from the periplasm [ Smith99 ]. When an outer membrane heme receptor is expressed in E. coli, the dipeptide ABC transporter is also capable of transporting heme and the heme precursor, δ aminolevulinic acid, from the periplasm into the cytoplasm [ Letoffe06 ]. Binding of heme to purified DppA has been demonstrated [ Letoffe06 ].

DppA accumulates to high levels when grown in minimal media, but levels of DppA are reduced when the medium is supplemented with casamino acids [ Olson91 ]. DppA levels were decreased after 4 hours exposure to zinc stress [ Easton06 ] and in response to glucose limitation [ Wick01 ]. When grown in rich medium, gcvB deletion mutants had high constitutive expression of dppA compared to the parent strain [ Urbanowski00 ]. dppA expression is also repressed by PhoB during phosphate limitation [ Smith92b ].

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GroEL-GroES chaperonin complex

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The Escherichia coli chaperone protein GroEL (Hsp60) and its regulator GroES are necessary for the proper folding of certain proteins [Kusukawa89, Goloubinof, Martin91, Horwich93]. The crystal structure of GroEL has been determined to a resolution of 2.8 angstroms. GroEL forms an 800 kDa cylinder from two back-to-back heptameric rings of 57 kDa subunits [Braig94]. X-ray crystallography to a resolution of 3.0 angstroms has determined the structure of GroEL-GroES complexed with seven ADP molecules. Hydrophobic residues of GroEL lining the interior of the rings are responsible for protein binding. GroES forms a heptamer of 10 kDa subunits which bind to the cis ring of the GroEL complex, forming a lid on the chamber [Xu97, Donald05]. Binding of GroES creates a large dome-shaped cavity with a highly polar inner surface in which a non-native protein has the opportunity to fold into its native form [Xu97].

There are 85 cytosolic proteins identified by LC-MS/MS after purification that absolutely require GroEL for proper folding, 13 of which are essential. These substrates were enriched for the (βα)8 triosephosphate isomerase (TIM) barrel domain. There are another approximately 165 that are only partially dependent upon GroEL for proper folding [Kerner05]. Global aggregation of newly translated proteins is observed in a GroEL deficient strain of E. coli - approximately 300 mainly cytosolic proteins were identified by electrophoretic analyses [Charbon11]. Sequence analysis has been performed to determine possible substrate recognition sequences for GroEL binding [Chaudhuri05].

GroEL and GroES are both heat inducible but are also expressed constitutively and are required for growth under all conditions tested [Fayet89]. In groEL temperature-sensitive mutants, a defined group of cytoplasmic proteins--including citrate synthase, ketoglutarate dehydrogenase, and polynucleotide phosphorylase--were translated but failed to reach native form [Horwich93]. In vivo studies were conducted and it was determined that overproduction of either GroEL and GroES or DnaK and DnaJ prevents aggregation of misfolded proteins. From these studies, it was proposed that GroEL and GroES proteins and the DnaK and DnaJ proteins have complementary functions in the folding and assembly of most proteins [Gragerov92]. Cellular localization studies, [Gaitanaris94], found that although GroEL does associate transiently with newly synthesized proteins, it is absent from the ribosomes. This suggests that DnaK and DnaJ play an early role in protein maturation, whereas GroEL acts at a later stage. GroEL is diffusely distributed under both normal and stress condtions [Charbon11].

Overexpressed GroEL/GroES promotes the folding of enzyme variants carrying mutations generated in vitro suggesting that it helps alleviate the destabilising constraints of protein mutations [Tokuriki09].

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ClpXP

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ClpXP is a serine protease complex responsible for the ATP-dependent degradation of a wide range of proteins [Gottesman93, Wojtkowiak93].
ClpXP degrades the altered Mu immunity repressor, Vir. When Vir is present, the normal immunity repressor, Rep, becomes more vulnerable to ClpXP-mediated degradation as well [Welty97]. ClpXP can also degrade MuA, although it does not degrade it all, allowing ClpX to act in its chaperone capacity to assist MuA function [Jones98, MhammediAl94].
ClpXP is partially responsible for degradation of proteins with the SsrA degradation tag, including SsrA-tagged lambda repressor [Bohn02, Gottesman98]. ClpXP degrades stably folded SsrA proteins efficiently, but only poorly degrades proteins bearing SsrA tags artificially attached in the middle of their sequences via cysteine linkages [Kenniston04].
ClpXP can degrade DNA-bound lambda O protein when transcription is possible, otherwise, it is stable [Zylicz98]. ClpXP-mediated degradation of lambda O protein can affect the lysis/lysogeny decision under certain growth conditions [Wojtkowiak93, Czyz01].
ClpXP is also required for degradation of the starvation-induced proteins Dps and sigma S during exponential growth [Stephani03, Schweder96].
Several other ClpXP substrates have been discovered. ClpXP degrades variants of the restriction enzyme EcoKI that have impaired enzymatic function, the mutagenically active protein UmuD' when it is in a heterodimer with unmodified UmuD and the antitoxin Phd from the Doc-Phd toxin/antitoxin pair (from plasmid prophage P1) [ONeill01, Frank96, Lehnherr95].
Putative ClpXP substrates were found by trapping with inactivated ClpP. Potential substrates included some with SsrA-like tails (crl, dksA, fnr, iscR, rplJ, rplU, gcp, pepB, katE, nrdH, tpx, chew, cysA, exbB, acnB, aldA, glpD, glyA, IldD and ycbW), MuA-like carboxy-terminal motifs (paaA, pncB, ribB, ybaQ), novel amino-terminal binding motifs (crl, dksA, fnr, lexA, rpoS, rplE, rplJ, rplK, rplS, rplU, tufB, dps, katE, nrdH, tpx, insH, chew, cysA, gatA, ompA, secA, aceA, atpD, cysD, dada, fabB, gapA, gatY, gatZ, glcB, glyA, iscS, lipA, moaA, pncB, tnaA, udp, ybaQ and ycbW) and no specific binding motifs (rseA, rplN, lon, clpX, dnaK, groEL, ftsZ, iscU, yebO and ygaT) [Flynn03].
SspB binds to SsrA-tagged proteins via its amino-terminal domain and enhances their degradation by ClpXP through carboxy-terminal binding to ClpXP [Levchenko00, Wah03]. SspB alone is sufficient to allow interaction with ClpXP. A protein that has been covalently linked to SspB becomes a ClpXP substrate even in the absence of an SsrA tag [Bolon04]. ClpX and SspB bind to overlapping parts of the SsrA tag, weakening the direct SsrA-ClpX interaction. The SspB-ClpX interaction overcomes this weakening effect [Hersch04]. Trapping experiments based on SspB show that RseA, which is cleaved from the membrane and binds to sigma E as an inhibitor during stress interacts with SspB and is degraded by ClpXP, thus releasing sigma E [Flynn04]. Sigma S degradation by ClpXP requires the adaptor RssB, which binds to Region 2.5 of sigma S, allowing binding of ClpX at the amino-terminus and subsequent degradation by ClpXP [Muffler96, Zhou01a, Studemann03]. Each ClpX hexamer has three SspB binding domains to match up with two ClpXP binding domains per SspB dimer, so only one SspB dimer can function with a given ClpX hexamer at a time [Bolon04a]. UmuD operates in a manner similar to SspB, binding to the ClpX amino-terminus and serving as a substrate tether for UmuD' [Neher03].
ClpXP consists of a ClpP tetradecamer capped at one or both ends by ClpX hexamers [Grimaud98].
Substrates bind to the distal surface of ClpX, and then are passed off to the inner cavity of ClpP to be degraded, in a process that is driven by ATP and modulated by ClpXP protease specificity-enhancing factor [Ortega00, Thibault06]. This process involves both static and dynamic contacts between ClpX and ClpP [Martin07]. The initial ClpX-mediated denaturation of substrate is the rate-limiting step in degradation of a well-folded protein, such as SsrA-tagged GFP [Kim00a].
ClpXP is required for limitation of lambda phage early DNA replication during slow growth [Wegrzyn].
ClpXP is required for acquisition of the genes encoding the restriction enzymes EcoKI and EcoAI by conjugation or transformation [Makovets98].
Despite lambda O initiator protein being a ClpXP substrate, lambda replication does not depend on ClpXP levels [Szalewska94].
Subunit composition of ClpXP = [(ClpP)14][(ClpX)6]2
ClpP serine protease = (ClpP)14
ClpX ATP-dependent protease specificity component and chaperone = (ClpX)6
Credits:
Last-Curated ? 09-Jan-2006 by Shearer A , SRI International
Enzymatic reaction of: protease (ClpXP)
a protein <=> a peptide + a peptide
The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.
Reversibility of this reaction is unspecified.

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ClpAP

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ClpAP is a serine protease complex responsible for the ATP-dependent degradation of a number of proteins [Katayama88]. Substrates for ClpAP include the plasmid P1 replication intiator RepA, HemA and a number of carbon starvation proteins [Wickner94, Wang99b, Damerau93]. ClpAP is also one of the proteases responsible for degradation of proteins tagged with the SsrA degradation marker, including tagged lambda repressor and tagged GFP (the latter substrate indicating that ClpAP can unfold stable, native protein in an ATP-dependent manner) [Gottesman98, WeberBan99]. ClpAP degrades a number of substrates that are not degraded by ClpXP [Gottesman93]. ClpAP is also responsible for rapid degradation of N-end rule substrates, which are marked for degradation by the identity of their amino-terminal residue (arginine, lysine, leucine, phenylalanine, tyrosine and tryptophan all mark a protein for N-end rule degradation) [Tobias91].
ClpAP consists of a ClpP tetradecamer capped at one or both ends by ClpA hexamers [Kessel95, Ishikawa]. The formation of this complex requires ATP binding and hydrolysis [Thompson94, Seol95, Maurizi98]. ATP is also required for degradation of larger polypeptide substrates by ClpAP [Thompson94]. ClpAP remains together as a complex through repeated rounds of degradation [Singh99]. ClpAP substrates interact with an allosteric site on ClpA prior to proteolysis by ClpP [Thompson94a].
A putative internal translation site variant of ClpA inhibits the interaction of full-length ClpA with ClpP, preventing formation of ClpAP [Seol94].
ClpS binds to the amino-terminal domain of ClpA, inhibiting degradation of SsrA-tagged proteins and of ClpA but accelerating disaggregation and degradation of heat-aggregated proteins in vitro [Dougan02, Zeth02].
ClpA levels increase during late exponential and early stationary phase, resulting in an increase in ClpAP activity [Katayama90].
ClpAP is required to maintain translation of the DNA protection protein Dps during starvation [Stephani03].
Subunit composition of ClpAP = [(ClpP)14][(ClpA)6]2
ClpP serine protease = (ClpP)14 ClpA ATP-dependent protease specificity component and chaperone = (ClpA)6