Aquifex aeolicus Aspartate Transcarbamoylase, an Enzyme Specialized for the Efficient Utilization of Unstable Carbamoyl Phosphate at Elevated Temperature

Purcarea, Cristina; Ahuja, Anupama; Lu, Tun; Kovari, Ladislau C.; Guy, Hedeel I.; Evans, David R.

doi:10.1074/jbc.m309383200

Cited by 38 publications

(57 citation statements)

References 45 publications

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“…The recombinant protein was purified by Ni 2ϩ affinity chromatography (23). A. aeolicus ATCase (22) and the CPSase GLN, CPS.A, and CPS.B subunits (21) were expressed and purified as previously described.…”

Section: Methodsmentioning

confidence: 99%

“…The isolated recombinant ATCase is fully active and forms transient complexes with the GLN⅐CPS.A⅐CPS.B (22). To determine whether DHOase or CPSase influences the function of the A. aeolicus ATCase, a similar series of titrations (Fig.…”

Section: Characterization Of a Aeolicus Dhoase-a Aeolicusmentioning

confidence: 99%

“…1). The CPSase (21) is a heterotrimeric protein consisting of one copy of an amidotransferase subunit (GLN) and two synthetase subunits (CPS.A and CPS.B), whereas ATCase (22) has been isolated as an active homotrimer. The DHOase from A. aeolicus has been classified (3) on the basis of its size and sequence as a type I DHOase.…”

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confidence: 99%

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Aquifex aeolicus Dihydroorotase

Ahuja¹,

Purcarea

Ebert

et al. 2004

Journal of Biological Chemistry

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Dihydroorotase (DHOase) catalyzes the reversible condensation of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis. The enzyme from Aquifex aeolicus, a hyperthermophilic organism of ancient lineage, was cloned and expressed in Escherichia coli. The purified protein was found to be a 45-kDa monomer containing a single zinc ion. Although there is no other DHOase gene in the A. aeolicus genome, the recombinant protein completely lacked catalytic activity at any temperature tested. However, DHOase formed an active complex with aspartate transcarbamoylase (ATCase) from the same organism. Whereas the k cat of 13.8 ؎ 0.03 s ؊1 was close to the value observed for the mammalian enzyme, the K m for dihydroorotate, 3.03 ؎ 0.05 mM was 433-fold higher. Gel filtration and chemical cross-linking showed that the complex exists as a 240-kDa hexamer (DHO 3 -ATC 3 ) and a 480-kDa duodecamer (DHO 6 -ATC 6 ) probably in rapid equilibrium. Complex formation protects both DHOase and ATCase against thermal degradation at temperatures near 100°C where the organism grows optimally. These results lead to the reclassification of both enzymes: ATCase, previously considered a Class C homotrimer, now falls into Class A, whereas the DHOase is a Class 1B enzyme. CD spectroscopy indicated that association with ATCase does not involve a significant perturbation of the DHOase secondary structure, but the visible absorption spectrum of a Co 2؉ -substituted DHOase is appreciably altered upon complex formation suggesting a change in the electronic environment of the active site. The association of DHOase with ATCase probably serves as a molecular switch that ensures that free, uncomplexed DHOase in the cell remains inactive. At pH 7.4, the equilibrium ratio of carbamoyl aspartate to dihydroorotate is 17 and complex formation may drive the reaction in the biosynthetic direction.

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Section: Methodsmentioning

confidence: 99%

Section: Characterization Of a Aeolicus Dhoase-a Aeolicusmentioning

confidence: 99%

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Aquifex aeolicus Dihydroorotase

Ahuja¹,

Purcarea

Ebert

et al. 2004

Journal of Biological Chemistry

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“…It is well known that CP is a critical metabolite for both arginine and pyrimidine biosynthesis, yet the short half-life of this molecule (t 1/2 Ͻ 2 s at 100°C) seems to necessitate some special mode of stabilization, especially in thermophilic organisms. Passing of metabolic intermediates between 2 enzymes that form permanent or transient complexes is one method of extending the lifetime of metabolic intermediates (6,7,16,17). However, the decomposition mechanism of the dianion of CP, which would exist under physiological conditions, proceeds through a unimolecular reaction involving an intramolecular COO bond breaking, thus a mechanism of protection must be more complex than a simple exclusion of water.…”

Section: Discussionmentioning

confidence: 99%

“…Hence, there must be an intracellular mechanism or mechanisms for the protection of CP from thermal decomposition. In the hyperthermophilic organism Aquifex aeolicus, channeling of CP between CP synthetase and ATCase has been established (7).…”

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confidence: 99%

Mechanism of thermal decomposition of carbamoyl phosphate and its stabilization by aspartate and ornithine transcarbamoylases

Wang

Xia

Guallar

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Carbamoyl phosphate (CP) has a half-life for thermal decomposition of <2 s at 100°C, yet this critical metabolic intermediate is found even in organisms that grow at 95-100°C. We show here that the binding of CP to the enzymes aspartate and ornithine transcarbamoylase reduces the rate of thermal decomposition of CP by a factor of >5,000. Both of these transcarbamoylases use an ordered-binding mechanism in which CP binds first, allowing the formation of an enzyme⅐CP complex. To understand how the enzyme⅐CP complex is able to stabilize CP we investigated the mechanism of the thermal decomposition of CP in aqueous solution in the absence and presence of enzyme. By quantum mechanics/molecular mechanics calculations we show that the critical step in the thermal decomposition of CP in aqueous solution, in the absence of enzyme, involves the breaking of the COO bond facilitated by intramolecular proton transfer from the amine to the phosphate. Furthermore, we demonstrate that the binding of CP to the active sites of these enzymes significantly inhibits this process by restricting the accessible conformations of the bound ligand to those disfavoring the reactive geometry. These results not only provide insight into the reaction pathways for the thermal decomposition of free CP in an aqueous solution but also show why these reaction pathways are not accessible when the metabolite is bound to the active sites of these transcarbamoylases.carbamoyltransferase ͉ quantum mechanics/molecular mechanics ͉ substrate stabilization ͉ thermophile C arbamoyl phosphate (CP) is a key metabolic intermediate in the biosynthesis of arginine and the pyrimidine nucleotides and for the detoxification of urea. In arginine biosynthesis, the enzyme ornithine (Orn) transcarbamoylase (OTCase) catalyzes the condensation of CP with Orn to form citrulline, an arginine precursor. In pyrimidine nucleotide biosynthesis, the enzyme aspartate transcarbamoylase (ATCase) catalyzes the condensation of CP and L-Asp to form N-carbamoyl-L-aspartate (CA).The quaternary structure of OTCase is a trimer of identical polypeptide chains. In organisms such as Bacillus subtilis, ATCase and OTCase have similar trimeric structures. The quaternary structure of ATCase is species-dependent; however, in all species the basic structural unit of the catalytic portion of ATCase is a trimer. The 3 active sites of the catalytic trimer of ATCases and OTCases are located at the subunit interfaces, and residues from the adjacent chain are necessary for catalysis. A sequence alignment of the domain responsible for the binding of CP in ATCases and OTCases suggests a common ancestral gene (1). The structural similarity of the CP domain from the two enzymes has been confirmed by X-ray crystallography (2, 3).CP is synthesized by the enzyme CP synthetase. This reaction proceeds via the highly unstable intermediates carboxyphosphate and carbamate. Escherichia coli CP synthetase has an internal tunnel between active sites that is proposed to channel these intermediates directly from one ac...

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Binding of Linear Anions and Formation of Anion Encapsulated Dimeric Capsular Assembly by Cis‐5,15‐bis(3,5‐trifluoromethylphenyl)calix[4]pyrrole

Patra,

Roy,

Gomila

et al. 2024

Eur J Org Chem

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Cis‐5,15‐bis(3,5‐trifluorometylphenyl)calix[4]pyrrole (1) forms stable 1/1 complexes with linear anions such as N3‐, SCN‑ and NCO‑ with the highest affinity for NCO‐ anion in acetonitrile solution. Crystallization of receptor 1 with TBASCN and (TMA)2SO4 resulted in the formation of dimeric capsular assembly, {(TMA)2[12•(SCN)2]}, in which two thiocyanate anions are encapsulated. On the other hand, 1•OCN(TMA) and 1•N3(TMA) complexes form one dimensional columnar structure in the solid state.

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Aquifex aeolicus Aspartate Transcarbamoylase, an Enzyme Specialized for the Efficient Utilization of Unstable Carbamoyl Phosphate at Elevated Temperature

Cited by 38 publications

References 45 publications

Aquifex aeolicus Dihydroorotase

Aquifex aeolicus Dihydroorotase

Mechanism of thermal decomposition of carbamoyl phosphate and its stabilization by aspartate and ornithine transcarbamoylases

Binding of Linear Anions and Formation of Anion Encapsulated Dimeric Capsular Assembly by Cis‐5,15‐bis(3,5‐trifluoromethylphenyl)calix[4]pyrrole

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