Allosteric regulation in Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase revisited: association of concerted homotropic cooperative interactions and local heterotropic effects

Tricot, Catherine; Villeret, Vincent; Sainz, G.; Dideberg, Otto; Stalon, Victor

doi:10.1006/jmbi.1998.2133

Cited by 15 publications

(15 citation statements)

References 22 publications

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“…Ornithine carbamoyltransferase (OTCase, Cthe_1869, 34 kDa) was identified at ~100 kDa, probably in a typical homo-trimer form [36-39]. Some studies suggest that OTCase is a cell surface protein [40,41] whereas Shi et al [42] reported that OTCase maybe a membrane-associated protein based on sequence analyses.…”

Section: Resultsmentioning

confidence: 99%

A Blue Native-PAGE analysis of membrane protein complexes in Clostridium thermocellum

Peng

Luo

et al. 2011

BMC Microbiol

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BackgroundClostridium thermocellum is a Gram-positive thermophilic anaerobic bacterium with the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. Identification and characterization of protein complexes in C. thermocellum are important toward understanding its metabolism and physiology.ResultsA two dimensional blue native/SDS-PAGE procedure was developed to separate membrane protein complexes of C. thermocellum. Proteins spots were identified by MALDI-TOF/TOF Mass spectrometry. 24 proteins were identified representing 13 distinct protein complexes, including several putative intact complexes. Interestingly, subunits of both the F1-F0-ATP synthase and the V1-V0-ATP synthase were detected in the membrane sample, indicating C. thermocellum may use alternative mechanisms for ATP generation.ConclusionTwo dimensional blue native/SDS-PAGE was used to detect membrane protein complexes in C. thermocellum. More than a dozen putative protein complexes were identified, revealing the simultaneous expression of two sets of ATP synthase. The protocol developed in this work paves the way for further functional characterization of these protein complexes.

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Section: Resultsmentioning

confidence: 99%

A Blue Native-PAGE analysis of membrane protein complexes in Clostridium thermocellum

Peng

Luo

et al. 2011

BMC Microbiol

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“…10 Conversely, anabolic OTCs (aOTCs) are cyclic homotrimers that display Michaelis-Menten kinetics. 5 These trimers are generally referred to as "catalytic trimers," as they constitute the basic catalytic structural unit in OTCs and, more generally, amongst the carbamoyltransferases. 11 Representative crystal structures of aOTCs from several sources have been determined: human aOTC complexed with the bisubstrate analog N-(phosphonacetyl)-Lornithine (PALO) 12 and more recently with CP and the inhibitor L-norvaline; 13 unliganded aOTC from Escherichia coli 14 as well as complexes with PALO 15 and with N δ -(N-sulfodiaminophosphinyl)-L-ornithine; 16 aOTC from Pyrococcus furiosus; 17 and, finally, aOTC from Mycobacterium tuberculosis, both unliganded and in complex with CP and L-norvaline.…”

Section: Introductionmentioning

confidence: 99%

“…As well as displaying homotropic cooperativity toward CP, this enzyme is allosterically activated by AMP or inorganic phosphate and inhibited by polyamines such as spermidine and putrescine. 5,7 Crystal structures of the native form of cOTC from P. aeruginosa 8,9 and a point mutant variant devoid of homotropic interactions 7 represent the only previously determined structures of a cOTC. The latter structure, in which Glu105 is substituted by Gly, reveals that the enzyme is composed of four trimers tetrahedrically arranged according to a 23 point group symmetry.…”

Section: Introductionmentioning

confidence: 99%

“…Although thermodynamically unfavored, 1 this chemical reaction is exploited by a number of microorganisms that degrade arginine because of the coupling with carbamate kinase, which catalyzes the next downstream step in the catabolic deiminase pathway and, in the case of cOTC from Pseudomonas aeruginosa, because of its low affinity toward CP and its strong cooperativity for this substrate. [2][3][4][5][6] Regulation of the enzymatic activity of cOTC from P. aeruginosa is highly complex. As well as displaying homotropic cooperativity toward CP, this enzyme is allosterically activated by AMP or inorganic phosphate and inhibited by polyamines such as spermidine and putrescine.…”

Section: Introductionmentioning

confidence: 99%

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Crystal Structure of the Hexameric Catabolic Ornithine Transcarbamylase from Lactobacillus hilgardii: Structural Insights into the Oligomeric Assembly and Metal Binding

Rivas

Fox

Ângulo

et al. 2009

Journal of Molecular Biology

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Catabolic ornithine transcarbamylase (cOTC; EC 2.1.3.3) catalyzes the formation of ornithine (ORN) and carbamoyl phosphate from citrulline, which constitutes the second step of the degradation of arginine via the arginine deiminase pathway. Here, we report the crystal structure of cOTC from the lactic acid bacteria Lactobacillus hilgardii (Lh-cOTC) refined to 2.1 A resolution. The structure reveals that Lh-cOTC forms a hexameric assembly, which was also confirmed by gel-filtration chromatography and analytical ultracentrifugation. The homohexamer, with 32 point group symmetry, represents a new oligomeric state within the members of the ornithine transcarbamylase family that are typically homotrimeric or homododecameric. The C-terminal end from each subunit constitutes a key structural element for the stabilization of the hexameric assembly in solution. Additionally, the structure reveals, for the first time in the ornithine transcarbamylase family, a metal-binding site located at the 3-fold molecular symmetry axis of each trimer.

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“…In other words, the allosteric interaction did not involve a “new” site, other than the active site, as is the case for M. tb PGDH. Allosteric substrate inhibition mechanisms have also been proposed for other enzymes such as tyrosine hydroxylase (14), ornithine carbamoyl transferase (15), and aspartate transcarbamylase (16). However, in these cases the identity of the allosteric site where substrate binds has not been determined.…”

Section: Discussionmentioning

confidence: 99%

Role of the Anion-Binding Site in Catalysis and Regulation of Mycobacterium tuberculosis d-3-Phosphoglycerate Dehydrogenase

Burton¹,

Chen²,

Xu³

et al. 2009

Biochemistry

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D-3-Phosphoglycerate dehydrogenase from M. tuberculosis displays substantial substrate inhibition by its physiological substrate in the direction of NADH oxidation, hydroxypyruvic acid phosphate (HPAP). Previous investigations showed that plots of substrate concentration versus activity derived from steady state assays could be fit with the equation for complete uncompetitive inhibition and that the mechanism may be allosteric. This investigation uses a simulation of transient kinetic data to demonstrate that the mechanism is consistent with the interaction of substrate at a second site called the anion-binding site. While substrate addition at the active site is ordered, with HPAP binding before NADH, NADH can compete with the substrate for binding to the allosteric site and thereby eliminate the substrate inhibition. Fluorescence resonance energy transfer analysis of mutants with specific tryptophan residues converted to phenylalanine residues demonstrates that the main interaction of NADH with the enzyme, in the absence of substrate, is at the allosteric anion-binding site. This is further confirmed by mutations of basic residues at the anion-binding site which also demonstrates that these residues are necessary for inhibition by L-serine when it binds to the regulatory domain. This may indicate that a ligand must be bound to the anion-binding site for Lserine inhibition, providing a potential mechanism for low levels of activity in the presence of high levels of inhibitor.D-3-phosphoglycerate dehydrogenase (PGDH 1 ) ( EC 1.1.1.95) from Mycobacterium tuberculosis (M. tb) is a homotetramer whose subunit's are identical in their primary structure but not in their tertiary structure. The crystal structure of M. tb PGDH (1) shows that the subunits are present in two different conformations that arise from a rotation about an interdomain connecting strand. Each subunit is made up of four distinct globular domains termed the nucleotide binding domain, the substrate binding domain, the intervening domain, and the regulatory domain. Residues 1-99 and 283-319 form the substrate binding domain, and residues 100-282 form the nucleotide binding domain. The intervening domain is made up of residues 320-454 and residues 455-529 complete the regulatory domain. The conformational rotation takes place in the strand that connects the substrate binding domain to the intervening domain. *Corresponding Author: Gregory A. Grant, Department of Medicine and of Developmental Biology, Washington University School of Medicine, St. Louis, As with E. coli PGDH (2), M. tb PGDH is inhibited by L-serine (3), which binds at the interface between adjacent regulatory domains in both enzymes. The regulatory domains are members of the ACT domain family (4) that are found in a large number of bacterial proteins mostly involved in amino acid metabolism or transcriptional regulation of amino acid biosynthesis.The intervening domain represents an additional domain not found in E. coli PGDH that "intervenes" between the substrate binding and ...

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Allosteric regulation in Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase revisited: association of concerted homotropic cooperative interactions and local heterotropic effects

Cited by 15 publications

References 22 publications

A Blue Native-PAGE analysis of membrane protein complexes in Clostridium thermocellum

A Blue Native-PAGE analysis of membrane protein complexes in Clostridium thermocellum

Crystal Structure of the Hexameric Catabolic Ornithine Transcarbamylase from Lactobacillus hilgardii: Structural Insights into the Oligomeric Assembly and Metal Binding

Role of the Anion-Binding Site in Catalysis and Regulation of Mycobacterium tuberculosis d-3-Phosphoglycerate Dehydrogenase

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