Characterization and assembly of the pseudomonas aeruginosa aspartate transcarbamoylase pseudo dihydroorotase complex. Aspartate transcarbamoylase is allosterically inhibited by the end. Atcase is the first enzyme unique to pyrimidine biosynthesis and a key enzyme for regulating purine, pyrimidine. Alba ruizramos, nada lallous, araceli grandegarcia and santiago ramonmaiques, expression, purification, crystallization and preliminary xray diffraction analysis of the aspartate transcarbamoylase domain of human cad, acta crystallographica section f structural biology and crystallization communications, 69, 12, 1425, 20. Pdf a new paradigm for allosteric regulation of escherichia coli. May 15, 2012 xray crystallography and smallangle xray scattering saxs in solution have been used to show that a mutant aspartate transcarbamoylase exists in an intermediate quaternary structure between the canonical t and r structures. Abstract abstract quorum sensing is the regulation of gene expression in response to fluctuations in cellpopulation density.
Kinetics and smallangle xray scattering experiments showed that pali can induce the cooperative. Aspartate transcarbamoylase atcase aspartate carbamoyltransferase, a key enzyme in pyrimidine biosynthesis gives a good instance of allosteric regulation. Bacterial atcases are divided into three classes, a, b and c. Aspartate carbamoyltransferase an overview sciencedirect. Pdf the catalytic and regulatory properties of aspartate. Proceedings of the national academy of sciences 2012, 109 20, 77417746. Revisiting the allosteric mechanism of aspartate transcarbamoylase.
Studies of trigonal and tetragonal crystalline forms of aspartate transcarbamylase show that the molecule has a threefold and a twofold symmetry axis. Aspartate carbamoyltransferase definition of aspartate. Aspartate transcarbamoylase is allosterically inhibited by. Comparative biochemistry and evolution of aspartate. Through structural and biochemical analyses, we revealed that r167s loop region in. Aspartate carbamoyltransferase also known as aspartate transcarbamoylase or atcase catalyzes the first step in the pyrimidine biosynthetic pathway ec 2. Allostery and cooperativity in escherichia coli aspartate transcarbamoylase.
Escherichia coli aspartate transcarbamoylase atcase catalyzes the committed step in pyrimidine nucleotide biosynthesis, the reaction between carbamoyl phosphate cp and l aspartate to form ncarbamoyll aspartate and inorganic phosphate. Characterization of the aspartate transcarbamoylase. The aspartate transcarbamoylase frombacillus subtilis, which lacks both homotropic and heterotropic properties, is an example of this class. The structure of aspartate transcarbamylase atcase. Cytidine triphosphate, an end product of the pyrimidine synthesis pathway, inhibits aspartate transcarbamoylase despite having little. The structures of the dihydroorotase dho and aspartate transcarbamoylase atc domains of human cad have been previously determined, but we lack information on how. The enzyme exhibits homotropic cooperativity and is allosterically regulated. Analysis of the atcase catalysis within the amino acid metabolism of the. Aug 17, 2011 malaria remains one of the leading causes of deaths attributable to a communicable disease globally. University of groningen analysis of the atcase catalysis.
The isolated catalytic subunit of atcase, which lacks the cooperative kinetic prop erties of the holoenyzme, exhibits only a very slight degree of cooperativity in. Aspartate carbamoyltransferase of plasmodium falciparum as a. This reaction is the first committed step of the pyrimidine biosynthetic pathway. Stably transfected mammalian cells expressing high levels of cad were. Aspartate transcarbamoylase stepthe second main source of pyrimidine ring aspartate combines with carbamoyl phosphate in the presence of aspartate transcarbamoylase. Aspartate transcarbamoylase atcase atcase carbamoyl phosphate carbamoyl aspartate aspartate pi ctp multiple reactions accumulating ctp inhibits enzyme cells in a high energy state have lots of atp atp activates atcase cells with abundant amino acids have lots of aspartate activates atcase. The structure of aspartate transcarbamylase atcase the structure of aspartate transcarbamylase, or atcase is described in this video. The regulation of carbamoyl phosphate synthetaseaspartate.
Aspartate transcarbamoylase assaysenzyme activity was deter mined according to the procedure of kempe et al. Aspartate transcarbamylaseof the 81 structures deposited in the pdb, 64 are of e. Pdf characterization and assembly of the pseudomonas. Comparative modeling of mammalian aspartate transcarbamylase. Aspartate transcarbamoylase atcase is a cytosolic enzyme that catalyzes the condensation of l aspartate and carbamoyl phosphate cp to produce ncarbamoyll aspartate caa. Trapping and structure determination of an intermediate in. Nphosphonacetyllisoasparagine a potent and specific. The aspartate transcarbamoylase atcase was purified from burkholderia cepacia 25416. Additionally, the saxs data indicate a phdependent structural alteration consistent with either a phinduced conformational change or a phinduced alteration in the t.
Cell extracts, stored at 70 c until all samples for a particular experiment had been collected, were assayed for enzyme activity and protein concentration at the same time. Aspartate carbamoyltransferase an overview sciencedirect topics. The enzyme aspartate transcarbamoylase atcase is a classic case of. Molecular weight determinations of the regulatory and catalytic chains and the aminoacid sequence of the r chain indicate that the atcase molecule contains six copies of each chain. Ijms free fulltext conformational plasticity of the. The cad gene encodes a trifunctional protein which is associated with the enzymatic activity of the first 3 enzymes in the 6step pathway of pyrimidine biosynthesis. Characterization of an aspartate transcarbamoylase cdna from pea pisum sativum l. Complex of nphosphonacetyll aspartate with aspartate carbamoyltransferase. In prokaryotic cells aspartate transcarbamoylase, an allosteric protein, is inhibited by the end product ctp and activated by.
Heterotropic interactions in aspartate transcarbamoylase pnas. Ctp inhibition of escherichia coli aspartate transcarbamoylase. Human atcase is a catalytic homotrimer with three cooperative active sites. Crystallographic determination of symmetry of aspartate.
Aspartate transcarbamylase atcase of escherichia coli. Pdf revisiting the allosteric mechanism of aspartate. Aspartate transcarbamoylase atcase has been studied for decades and escherichia coli atcase is referred as a textbook example for both feedback regulation and cooperativity. Other than for strictly personal use, it is not permitted to download or to. Xray refinement, analysis of conformational changes and catalytic and allosteric mechanisms. Structure and functional characterization of human aspartate. In the course of purification, three different atcase activities appeared namely dodecameric 550 kda holoenzyme, and two trimeric atcases of 140 kda consists of 47 kda pyrb subunits and 120 kda consists of 40 kda pyrb subunits each. Genes encoding escherichia coli aspartate transcarbamoylase. The regulation of carbamoyl phosphate synthetase aspartate transcarbamoylase dihydroorotase cad by phosphorylation and proteinprotein interactions.
The reemergence of drugresistant plasmodium falciparum, the most fatal human malarial parasite, has necessitated the exploration of different pathways to provide the urgently required novel drug targets. However, several critical questions about the catalytic and regulatory mechanisms of e. Characterization of an aspartate transcarbamoylase cdna from. Aspartate transcarbamoylase atcase catalyzes the first committed step in pyrimidine biosynthesis. In prokaryotes, the first three enzymes in pyrimidine biosynthesis, carbamoyl phosphate synthetase cps, aspartate transcarbamoylase atc, and dihydroorotase dho, are commonly expressed separately and either function independently escherichia co. The 120 kda pyrb polypeptide arose by specific cleavage of the pyrb. The reaction catalyzed by this enzyme is the condensation of aspartate and carbamoyl phosphate to form ncarbamoylaspartate and orthophosphate figure 10. Escherichia coli aspartate transcarbamoylaseatcase. This gene encodes a trifunctional protein which is associated with the enzymatic activities of the first 3 enzymes in the 6step pathway of pyrimidine biosynthesis. The synthesis of a new inhibitor, nphosphonacetyllisoasparagine pali, of escherichia coli aspartate transcarbamoylase atcase is reported, as well as structural studies of the enzymepali complex.
Allosteric regulation of enzymes metabolic pathways do not run on a continuous basis, but are regulated according to need catabolic pathways run if there is demand for atp. New mechanismbased inhibitors of aspartate transcarbamoylase. Trapping and structure determination of an intermediate in the allosteric transition of aspartate transcarbamoylase. Multiple activities of aspartate transcarbamoylase in. Quorum sensing bacteria produce and release chemical signal molecules called autoinducers that increase in concentration as a function. This step is the committed step of the pathway as this enzyme is allosterically regulated allosteric inhibition by ctp. A second form of aspartate transcarbamoylase, class a, is a dodecamer of six 34kda and six 45kda polypeptides. Cad, the multifunctional protein controlling the synthesis of pyrimidines, associates through its aspartate transcarbamoylase domain atcase into 1.
Aspartate transcarbamoylase catalyzes the first step in the biosynthesis of pyrimidines, bases that are components of nucleic acids. The enzyme undergoes as it shifts between its lowactivity, lowaffinity form, t state, to its highactivity, highaffinity form, r state, and allosteric effectors modulate the activity. Substitutions in the aspartate transcarbamoylase domain of. Insights into the mechanisms of catalysis and heterotropic regulation of escherichia coli aspartate transcarbamoylase based upon a structure of the enzyme complexed with the bisubstrate analogue nphosphonacetyll aspartate at 2. The overlapping pyrbc genes encode the pseudomonases atcase, which is active only as a 480 kda dodecamer and requires an inactive pyrc.
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