The inhibition of ornithine transcarbamoylase from Escherichia coli W by phaseolotoxin.
Ontology highlight
ABSTRACT:
SUBMITTER: Templeton MD
PROVIDER: S-EPMC1144443 | biostudies-other | 1984 Dec
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Understanding how enzymes achieve their tremendous catalytic power is a major question in biochemistry. Greater understanding is also needed for enzyme engineering applications. In many cases, enzyme efficiency and specificity depend on residues not in direct contact with the substrate, termed remote residues. This work focuses on Escherichia coli ornithine transcarbamoylase (OTC), which plays a central role in amino acid metabolism. OTC has been reported to undergo an induced-fit conformational change upon binding its first substrate, carbamoyl phosphate (CP), and several residues important for activity have been identified. Using computational methods based on the computed chemical properties from theoretical titration curves, sequence-based scores derived from evolutionary history, and protein surface topology, residues important for catalytic activity were predicted. The roles of these residues in OTC activity were tested by constructing mutations at predicted positions, followed by steady-state kinetics assays and substrate binding studies with the variants. First-layer mutations R57A and D231A, second-layer mutation H272L, and third-layer mutation E299Q, result in 57- to 450-fold reductions in kcat/KM with respect to CP and 44- to 580-fold reductions with respect to ornithine. Second-layer mutations D140N and Y160S also reduce activity with respect to ornithine. Most variants had decreased stability relative to wild-type OTC, with variants H272L, H272N, and E299Q having the greatest decreases. Variants H272L, E299Q, and R57A also show compromised CP binding. In addition to direct effects on catalytic activity, effects on overall protein stability and substrate binding were observed that reveal the intricacies of how these residues contribute to catalysis.
| S-EPMC7004355 | biostudies-literature
Project description:Enzymes catalyze a particular reaction in cells, but only a few control the rate of this reaction and the metabolic pathway that follows. One specific mechanism for such enzymatic control of a metabolic pathway involves molecular feedback, whereby a metabolite further down the pathway acts at a unique site on the control enzyme to alter its activity allosterically. This regulation may be positive or negative (or both), depending upon the particular system. Another method of enzymatic control involves the cooperative binding of the substrate, which allows a large change in enzyme activity to emanate from only a small change in substrate concentration. Allosteric regulation and homotropic cooperativity are often known to involve significant conformational changes in the structure of the protein. Escherichia coli aspartate transcarbamoylase (ATCase) is the textbook example of an enzyme that regulates a metabolic pathway, namely, pyrimidine nucleotide biosynthesis, by feedback control and by the cooperative binding of the substrate, L-aspartate. The catalytic and regulatory mechanisms of this enzyme have been extensively studied. A series of X-ray crystal structures of the enzyme in the presence and absence of substrates, products, and analogues have provided details, at the molecular level, of the conformational changes that the enzyme undergoes as it shifts between its low-activity, low-affinity form (T state) to its high-activity, high-affinity form (R state). These structural data provide insights into not only how this enzyme catalyzes the reaction between l-aspartate and carbamoyl phosphate to form N-carbamoyl-L-aspartate and inorganic phosphate, but also how the allosteric effectors modulate this activity. In this Account, we summarize studies on the structure of the enzyme and describe how these structural data provide insights into the catalytic and regulatory mechanisms of the enzyme. The ATCase-catalyzed reaction is regulated by nucleotide binding some 60 Å from the active site, inducing structural alterations that modulate catalytic activity. The delineation of the structure and function in this particular model system will help in understanding the molecular basis of cooperativity and allosteric regulation in other systems as well.
| S-EPMC3276696 | biostudies-literature
Project description:The allosteric enzyme aspartate transcarbamoylase (ATCase) from Escherichia coli has been the subject of investigations for approximately 50 years. This enzyme controls the rate of pyrimidine nucleotide biosynthesis by feedback inhibition, and helps to balance the pyrimidine and purine pools by competitive allosteric activation by ATP. The catalytic and regulatory components of the dodecameric enzyme can be separated and studied independently. Many of the properties of the enzyme follow the Monod, Wyman Changeux model of allosteric control thus E. coli ATCase has become the textbook example. This review will highlight kinetic, biophysical, and structural studies which have provided a molecular level understanding of how the allosteric nature of this enzyme regulates pyrimidine nucleotide biosynthesis.
| S-EPMC3393099 | biostudies-literature
Project description:Escherichia coli aspartate transcarbamoylase is feedback inhibited by CTP and UTP in the presence of CTP. Here, we show by X-ray crystallography that UTP binds to a unique site on each regulatory chain of the enzyme that is near but not overlapping with the known CTP site. These results bring into question all of the previously proposed mechanisms of allosteric regulation in aspartate transcarbamoylase.
| S-EPMC3428212 | biostudies-literature
Project description:Here we report the isolation, kinetic characterization, and X-ray structure determination of a cooperative Escherichia coli aspartate transcarbamoylase (ATCase) without regulatory subunits. The native ATCase holoenzyme consists of six catalytic chains organized as two trimers bridged noncovalently by six regulatory chains organized as three dimers, c(6)r(6). Dissociation of the native holoenzyme produces catalytically active trimers, c(3), and nucleotide-binding regulatory dimers, r(2). By introducing specific disulfide bonds linking the catalytic chains from the upper trimer site specifically to their corresponding chains in the lower trimer prior to dissociation, a new catalytic unit, c(6), was isolated consisting of two catalytic trimers linked by disulfide bonds. Not only does the c(6) species display enhanced enzymatic activity compared to the wild-type enzyme, but the disulfide bonds also impart homotropic cooperativity, never observed in the wild-type c(3). The c(6) ATCase was crystallized in the presence of phosphate and its X-ray structure determined to 2.10 A resolution. The structure of c(6) ATCase liganded with phosphate exists in a nearly identical conformation as other R-state structures with similar values calculated for the vertical separation and planar angles. The disulfide bonds linking upper and lower catalytic trimers predispose the active site into a more active conformation by locking the 240s loop into the position characteristic of the high-affinity R state. Furthermore, the elimination of the structural constraints imposed by the regulatory subunits within the holoenzyme provides increased flexibility to the c(6) enzyme, enhancing its activity over the wild-type holoenzyme (c(6)r(6)) and c(3). The covalent linkage between upper and lower catalytic trimers restores homotropic cooperativity so that a binding event at one or so active sites stimulates binding at the other sites. Reduction of the disulfide bonds in the c(6) ATCase results in c(3) catalytic subunits that display kinetic parameters similar to those of wild-type c(3). This is the first report of an active c(6) catalytic unit that displays enhanced activity and homotropic cooperativity.
| S-EPMC2935174 | biostudies-literature
Project description:The activity and cooperativity of Escherichia coli aspartate transcarbamoylase (ATCase) vary as a function of pH, with a maximum of both parameters at approximately pH 8.3. Here we report the first X-ray structure of unliganded ATCase at pH 8.5, to establish a structural basis for the observed Bohr effect. The overall conformation of the active site at pH 8.5 more closely resembles the active site of the enzyme in the R-state structure than other T-state structures. In the structure of the enzyme at pH 8.5 the 80's loop is closer to its position in R-state structures. A unique electropositive channel, comprised of residues from the 50's region, is observed in this structure, with Arg54 positioned in the center of the channel. The planar angle between the carbamoyl phosphate and aspartate domains of the catalytic chain is more open at pH 8.5 than in ATCase structures determined at lower pH values. The structure of the enzyme at pH 8.5 also exhibits lengthening of a number of interactions in the interface between the catalytic and regulatory chains, whereas a number of interactions between the two catalytic trimers are shortened. These alterations in the interface between the upper and lower trimers may directly shift the allosteric equilibrium and thus the cooperativity of the enzyme. Alterations in the electropositive environment of the active site and alterations in the position of the catalytic chain domains may be responsible for the enhanced activity of the enzyme at pH 8.5.
| S-EPMC2920061 | biostudies-literature
Project description:The crystal structure of Escherichia coli ornithine transcarbamoylase (OTCase, EC 2.1.3.3) complexed with the bisubstrate analog N-(phosphonacetyl)-L-ornithine (PALO) has been determined at 2.8-A resolution. This research on the structure of a transcarbamoylase catalytic trimer with a substrate analog bound provides new insights into the linkages between substrate binding, protein-protein interactions, and conformational change. The structure was solved by molecular replacement with the Pseudomonas aeruginosa catabolic OTCase catalytic trimer (Villeret, V., Tricot, C., Stalon, V. & Dideberg, O. (1995) Proc. Natl. Acad. Sci. USA 92, 10762-10766; Protein Data Bank reference pdb 1otc) as the model and refined to a crystallographic R value of 21.3%. Each polypeptide chain folds into two domains, a carbamoyl phosphate binding domain and an L-ornithine binding domain. The bound inhibitor interacts with the side chains and/or backbone atoms of Lys-53, Ser-55, Thr-56, Arg-57, Thr-58, Arg-106, His-133, Asn-167, Asp-231, Met-236, Leu-274, Arg-319 as well as Gln-82 and Lys-86 from an adjacent chain. Comparison with the unligated P. aeruginosa catabolic OTCase structure indicates that binding of the substrate analog results in closure of the two domains of each chain. As in E. coli aspartate transcarbamoylase, the 240s loop undergoes the largest conformational change upon substrate binding. The clinical implications for human OTCase deficiency are discussed.
| S-EPMC23215 | biostudies-literature
Project description:Escherichia coli aspartate transcarbamoylase (ATCase) allosterically regulates pyrimidine nucleotide biosynthesis. The enzyme is inhibited by CTP and can be further inhibited by UTP, although UTP alone has little or no influence on activity; however, the mechanism for the synergistic inhibition is still unknown. To determine how UTP is able to synergistically inhibit ATCase in the presence of CTP, we determined a series of X-ray structures of ATCase·nucleotide complexes. Analysis of the X-ray structures revealed that (1) CTP and dCTP bind in a very similar fashion, (2) UTP, in the presence of dCTP or CTP, binds at a site that does not overlap the CTP/dCTP site, and (3) the triphosphates of the two nucleotides are parallel to each other with a metal ion, in this case Mg(2+), coordinated between the ?- and ?-phosphates of the two nucleotides. Kinetic experiments showed that the presence of a metal ion such as Mg(2+) is required for synergistic inhibition. Together, these results explain how the binding of UTP can enhance the binding of CTP and why UTP binds more tightly in the presence of CTP. A mechanism for the synergistic inhibition of ATCase is proposed in which the presence of UTP stabilizes the T state even more than CTP alone. These results also call into question many of the past kinetic and binding experiments with ATCase with nucleotides as the presence of metal contamination was not considered important.
| S-EPMC3461825 | biostudies-literature
Project description:Allosteric regulation of key metabolic enzymes is a fascinating field to study the structure-function relationship of induced conformational changes of proteins. In this review we compare the principles of allosteric transitions of the complex classical model aspartate transcarbamoylase (ATCase) from Escherichia coli, consisting of 12 polypeptides, and the less complicated chorismate mutase derived from baker's yeast, which functions as a homodimer. Chorismate mutase presumably represents the minimal oligomerization state of a cooperative enzyme which still can be either activated or inhibited by different heterotropic effectors. Detailed knowledge of the number of possible quaternary states and a description of molecular triggers for conformational changes of model enzymes such as ATCase and chorismate mutase shed more and more light on allostery as an important regulatory mechanism of any living cell. The comparison of wild-type and engineered mutant enzymes reveals that current textbook models for regulation do not cover the entire picture needed to describe the function of these enzymes in detail.
| S-EPMC99034 | biostudies-literature
Project description:Escherichia coli is a common host for biotechnology and synthetic biology applications. During growth and fermentation, the microbes are often exposed to stress conditions, such as variations in pH or solvent concentrations. Bacterial membranes play a key role in response to abiotic stresses. Ornithine lipids (OLs) are a group of membrane lipids whose presence and synthesis have been related to stress resistance in bacteria. We wondered if this stress resistance could be transferred to bacteria not encoding the capacity to form OLs in their genome, such as E. coli. In this study, we engineered different E. coli strains to produce unmodified OLs and hydroxylated OLs by expressing the synthetic operon olsFC. Our results showed that OL formation improved pH resistance and increased biomass under phosphate limitation. Transcriptome analysis revealed that OL-forming strains differentially expressed stress- and membrane-related genes. OL-producing strains also showed better growth in the presence of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), suggesting reduced proton leakiness in OL-producing strains. Furthermore, our engineered strains showed improved heterologous violacein production at phosphate limitation and also at low pH. Overall, this study demonstrates the potential of engineering the E. coli membrane composition for constructing robust hosts with an increased abiotic stress resistance for biotechnology and synthetic biology applications. KEY POINTS: • Ornithine lipid production in E. coli increases biomass yield under phosphate limitation. • Engineered strains show an enhanced production phenotype under low pH stress. • Transcriptome analysis and CCCP experiments revealed reduced proton leakage.
| S-EPMC11001654 | biostudies-literature