Genes encoding escherichia coli aspartate transcarbamoylase. 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. 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. Through structural and biochemical analyses, we revealed that r167s loop region in. Aug 17, 2011 malaria remains one of the leading causes of deaths attributable to a communicable disease globally. 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. Heterotropic interactions in aspartate transcarbamoylase pnas. Aspartate transcarbamoylase atcase is a cytosolic enzyme that catalyzes the condensation of l aspartate and carbamoyl phosphate cp to produce ncarbamoyll aspartate caa. Pdf revisiting the allosteric mechanism of aspartate. Complex of nphosphonacetyll aspartate with aspartate carbamoyltransferase. Aspartate transcarbamoylase atcase proteopedia, life in 3d.
However, several critical questions about the catalytic and regulatory mechanisms of e. Cad, the multifunctional protein controlling the synthesis of pyrimidines, associates through its aspartate transcarbamoylase domain atcase into 1. Aspartate transcarbamoylase assaysenzyme activity was deter mined according to the procedure of kempe et al. Pdf the catalytic and regulatory properties of aspartate. Aspartate carbamoyltransferase an overview sciencedirect. 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. Atcase is the first enzyme unique to pyrimidine biosynthesis and a key enzyme for regulating purine, pyrimidine. Cytidine triphosphate, an end product of the pyrimidine synthesis pathway, inhibits aspartate transcarbamoylase despite having little. Aspartate carbamoyltransferase definition of aspartate.
The aspartate transcarbamoylase atcase was purified from burkholderia cepacia 25416. Aspartate transcarbamoylase, regulation of enzyme activity. 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. An enzyme that catalyzes the conversion of carbamoyl phosphate and l aspartate to yield orthophosphate and ncarbamoyll aspartate from enzyme nomenclature, 1992 ec 2. 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. Trapping and structure determination of an intermediate in. Aspartate transcarbamylase atcase of escherichia coli. Characterization and assembly of the pseudomonas aeruginosa aspartate transcarbamoylase pseudo dihydroorotase complex. Aspartate transcarbamoylase atcase aspartate carbamoyltransferase, a key enzyme in pyrimidine biosynthesis gives a good instance of allosteric regulation. The regulation of carbamoyl phosphate synthetaseaspartate. A second form of aspartate transcarbamoylase, class a, is a dodecamer of six 34kda and six 45kda polypeptides. University of groningen analysis of the atcase catalysis.
Analysis of the atcase catalysis within the amino acid metabolism of the. 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. 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. This step is the committed step of the pathway as this enzyme is allosterically regulated allosteric inhibition by ctp. Comparative biochemistry and evolution of aspartate. 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. Aspartate transcarbamoylase is allosterically inhibited by. Aspartate transcarbamoylase stepthe second main source of pyrimidine ring aspartate combines with carbamoyl phosphate in the presence of aspartate transcarbamoylase. Aspartate transcarbamylaseof the 81 structures deposited in the pdb, 64 are of e. Aspartate carbamoyltransferase of plasmodium falciparum as a.
Escherichia coli aspartate transcarbamoylaseatcase. Nphosphonacetyllisoasparagine a potent and specific. Xray refinement, analysis of conformational changes and catalytic and allosteric mechanisms. Pdf the catalytic and regulatory properties of aspartate transcarbamoylase from pyrococcus abyssi were studied in the ge5 strain. The structure of aspartate transcarbamylase atcase. Aspartate transcarbamoylase atcase catalyzes the first committed step in pyrimidine biosynthesis. The overlapping pyrbc genes encode the pseudomonases atcase, which is active only as a 480 kda dodecamer and requires an inactive pyrc. Pdf a new paradigm for allosteric regulation of escherichia coli. Ctp inhibition of escherichia coli aspartate transcarbamoylase. Aspartate transcarbamoylase is allosterically inhibited by the end.
Characterization of the aspartate transcarbamoylase. The structure of aspartate transcarbamylase atcase the structure of aspartate transcarbamylase, or atcase is described in this video. Bacterial atcases are divided into three classes, a, b and c. The 120 kda pyrb polypeptide arose by specific cleavage of the pyrb. Additionally, the saxs data indicate a phdependent structural alteration consistent with either a phinduced conformational change or a phinduced alteration in the t. Aspartate carbamoyltransferase also known as aspartate transcarbamoylase or atcase catalyzes the first step in the pyrimidine biosynthetic pathway ec 2.
Aspartate carbamoyltransferase an overview sciencedirect topics. Allostery and cooperativity in escherichia coli aspartate transcarbamoylase. The aspartate transcarbamoylase frombacillus subtilis, which lacks both homotropic and heterotropic properties, is an example of this class. 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. Stably transfected mammalian cells expressing high levels of cad were. The structures of the dihydroorotase dho and aspartate transcarbamoylase atc domains of human cad have been previously determined, but we lack information on how. This reaction is the first committed step of the pyrimidine biosynthetic pathway. Studies of trigonal and tetragonal crystalline forms of aspartate transcarbamylase show that the molecule has a threefold and a twofold symmetry axis. Escherichia coli atcase is a prototypic enzyme regulated by both product feedback and substrate cooperativity, whereas human atcase is a potential anticancer target. New mechanismbased inhibitors of aspartate transcarbamoylase.
Revisiting the allosteric mechanism of aspartate transcarbamoylase. The atcase catalyzes the formation of ncarbamoylaspartate from aspartate and carbamoyl phosphate and is the committed step in pyrimidines biosynthesis in the figure. 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. 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. Comparative modeling of mammalian aspartate transcarbamylase.
Alanine substitutions for the atcase residues asp90 and arg269 were generated in a bicistronic vector that encodes a 6histidinetagged hamster cad. Abstract abstract quorum sensing is the regulation of gene expression in response to fluctuations in cellpopulation density. 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. Structure and functional characterization of human aspartate. In prokaryotic cells aspartate transcarbamoylase, an allosteric protein, is inhibited by the end product ctp and activated by. Proceedings of the national academy of sciences 2012, 109 20, 77417746. Quorum sensing bacteria produce and release chemical signal molecules called autoinducers that increase in concentration as a function. Ijms free fulltext conformational plasticity of the.
The enzyme exhibits homotropic cooperativity and is allosterically regulated. Substitutions in the aspartate transcarbamoylase domain of. Pdf characterization and assembly of the pseudomonas. Multiple activities of aspartate transcarbamoylase in. 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. 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. Aspartate transcarbamoylase atcase has been studied for decades and escherichia coli atcase is referred as a textbook example for both feedback regulation and cooperativity.
Characterization of an aspartate transcarbamoylase cdna from pea pisum sativum l. Trapping and structure determination of an intermediate in the allosteric transition of aspartate transcarbamoylase. The enzyme aspartate transcarbamoylase atcase is a classic case of. Kinetics and smallangle xray scattering experiments showed that pali can induce the cooperative. Crystallographic determination of symmetry of aspartate.
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