Purification of aspartate transcarbamoylase from<i>Pseudomonas syringae</i>

Shepherdson, Margaret; McPhail, Donald R.

doi:10.1111/j.1574-6968.1993.tb06574.x

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FEMS Microbiology Letters

1993

DOI: 10.1111/j.1574-6968.1993.tb06574.x

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Purification of aspartate transcarbamoylase fromPseudomonas syringae

Margaret Shepherdson

Donald R. McPhail

Abstract: The aspartate transcarbamoylase (ATCase) from Pseudomonas syringae has been purified. The purified enzyme was shown by SDS-PAGE to give two bands. Unambiguous results from N-terminal sequencing suggested that each band represented a homogeneous polypeptide. The M(r) (relative molecular mass) of the polypeptides was estimated to be 47 kDa and 34 kDa. The M(r) of the holoenzyme determined by gel filtration and electrophoretic migration in polyacrylamide gradient gels under non-denaturing conditions was estimated… Show more

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Cited by 5 publications

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“…This type of pseudo-dihydrooro-tase domain was subsequently found in several other organisms (16). For example, Pseudomonas ATCase is a duodecameric molecule consisting of six ATCase catalytic chains and six polypeptides (17)(18)(19) that are homologous to DHOase (20) but it lacks the zinc binding ligands and is inactive.…”

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confidence: 99%

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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mentioning

confidence: 99%

Aquifex aeolicus Dihydroorotase

Ahuja¹,

Purcarea

Ebert

et al. 2004

Journal of Biological Chemistry

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Pseudomonas aeruginosa Aspartate Transcarbamoylase

Vickrey¹,

Hervé²,

Evans³

2002

Journal of Biological Chemistry

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Aspartate transcarbamoylase from Pseudomonadaceae is a class A enzyme consisting of six copies of a 36-kDa catalytic chain and six copies of a 45-kDa polypeptide of unknown function. The 45-kDa polypeptide is homologous to dihydroorotase but lacks catalytic activity. Pseudomonas aeruginosa aspartate transcarbamoylase was overexpressed in Escherichia coli. The homogeneous His-tagged protein isolated in high yield, 30 mg/liter of culture, by affinity chromatography and crystallized. Attempts to dissociate the catalytic and pseudo-dihydroorotase (pDHO) subunits or to express catalytic subunits only were unsuccessful suggesting that the pDHO subunits are required for the proper folding and assembly of the complex. As reported previously, the enzyme was inhibited by micromolar concentrations of all nucleotide triphosphates. In the absence of effectors, the aspartate saturation curves were hyperbolic but became strongly sigmoidal in the presence of low concentrations of nucleotide triphosphates. The inhibition was unusual in that only free ATP, not MgATP, inhibits the enzyme. Moreover, kinetic and binding studies with a fluorescent ATP analog suggested that ATP induces a conformational change that interferes with the binding of carbamoyl phosphate but has little effect once carbamoyl phosphate is bound. The peculiar allosteric properties suggest that the enzyme may be a potential target for novel chemotherapeutic agents designed to combat Pseudomonas infection.Pseudomonadaceae is a family of eubacteria with a wide ecological distribution that is pathogenic in animals (1, 2). Pseudomonas aeruginosa, the principle cause of morbidity and mortality in immunocompromised patients and burn victims (3-5), is an obligate pathogen. In cystic fibrosis, P. aeruginosa infection is the usual cause of death. In the design of therapeutic strategies, comparatively little attention has focused on the physiology of the organism itself, despite the unusual metabolism exhibited by Pseudomonadaceae such as uracil catabolism (6), the lack of gene repression (7,8), and the ability to use arginine as the sole source of carbon, nitrogen, and energy (9, 10).In other organisms, studies of de novo pyrimidine biosynthesis and salvage are actively pursued because of the relationship of these pathways to growth, development, and chemotherapy. Aspartate transcarbamoylase (ATCase, 1 EC 2.1.3.2) catalyzes the formation of carbamoyl aspartate from carbamoyl phosphate and aspartate (11) in the de novo biosynthetic pathway. The structure and function of the enzyme from Escherichia coli has been extensively studied and has become the prototype of allosteric enzymes. However, ATCases are highly polymorphic differing both in structure and mode of regulation. Jones and co-workers (12) identified three distinct classes of bacterial aspartate transcarbamoylase.E. coli ATCase, designated a class B enzyme, consists of six copies of two types of polypeptide chains, catalytic and regulatory. The 34-kDa catalytic chains associate to form catalytically active but unr...

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A reappraisal of the diversity and class distribution of aspartate transcarbamoylases in Gram-negative bacteria

1996

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Purification of aspartate transcarbamoylase fromPseudomonas syringae

Cited by 5 publications

References 12 publications

Aquifex aeolicus Dihydroorotase

Aquifex aeolicus Dihydroorotase

Pseudomonas aeruginosa Aspartate Transcarbamoylase

A reappraisal of the diversity and class distribution of aspartate transcarbamoylases in Gram-negative bacteria

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