Localization of the site for the nucleotide effectors of<i>Escherichia coli</i>carbamoyl phosphate synthetase using site‐directed mutagenesis

Mora, Paz; Rubio, Vicente; Fresquet, Vicente; Cervera, Javier

doi:10.1016/s0014-5793(99)00197-0

Cited by 16 publications

(14 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…IMP binding has been localized to domain D by photolabeling (33), site-directed mutagenesis (34 -39), and crystal structure analysis (28). UMP binding has also been localized to domain D by photolabeling (40) and site-directed mutagenesis (35)(36)(37)(38)(39). Studies with a number of UMP and IMP 2 Primers (with mutated residues in bold): P1, 5Ј-AAGTACCAACT-AGTTCCATGTACC-3Ј; P2, 5Ј-GGCCGCTCTAAAGCTTGTGGATCC-3Ј; P3, 5Ј-GGGCAGCAACTAGTCCATGAAG-3Ј; P4, 5Ј-CTTCATGGA-CTAGTTGCTGCCC-3Ј; P5, 5Ј-GCAGCTGGGCAGCAACAACTTCCAT-GTACCACTACC-3Ј; P6, 5Ј-GGTAGTGGTACATGGAAGTTGT-TGCTGCCCAGCTGC-3Ј.…”

Section: Resultsmentioning

confidence: 99%

“…The chCPS response to ornithine was not surprising because ornithine is known to bind at the domain C/D interface (29,30,35,36,42,43), and because domain C of chCPS is derived from the ornithineresponsive eCPS. However, the chCPS response to UMP was unexpected because the binding site for pyrimidine nucleotides resides entirely in domain D (29,30,(35)(36)(37)(38)(39)(40). We therefore screened a second pyrimidine nucleotide, UTP, which is known to inhibit eCPS but only at 100 times higher concentrations than required for UMP (3).…”

Section: Screening Of Potential Allosteric Effectors With Chcps-mentioning

confidence: 99%

“…Binding of Allosteric Effectors to eCPS, sCPS, and chCPSFor eCPS, domain D is known to be the sole (IMP/UMP) or partial (ornithine) locus for binding allosteric effectors (29,30,(33)(34)(35)(36)(37)(38)(39)(40)(41)(42). Occupancy of the effector site is then communicated principally to the ATP C active site and secondarily to the ATP B active site (1).…”

Section: Screening Of Potential Allosteric Effectors With Chcps-mentioning

confidence: 99%

See 2 more Smart Citations

Unmasking a Functional Allosteric Domain in an Allosterically Nonresponsive Carbamoyl-phosphate Synthetase

Eroglu

Powers‐Lee

2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

Although carbamoyl-phosphate synthetases (CPSs) share sequence identity, multidomain structure, and reaction mechanism, they have varying physiological roles and allosteric effectors. Escherichia coli CPS (eCPS) provides CP for both arginine and pyrimidine nucleotide biosynthesis and is allosterically regulated by metabolites from both pathways, with inhibition by UMP and activation by IMP and ornithine. The argininespecific CPS from Saccharomyces cerevisiae (sCPS), however, apparently responds to no allosteric effectors. We have designed and analyzed a chimeric CPS (chCPS, in which the C-terminal 136 residues of eCPS were replaced by the corresponding residues of sCPS) to define the structural basis for the allosteric nonresponsiveness of sCPS and thereby provide insight into the mechanism for allosteric selectivity and responsiveness in the other CPSs. Surprisingly, ornithine and UMP each had a significant effect on chCPS activity, and did so at concentrations that were similar to those effective for eCPS. We further found that sCPS bound both UMP and IMP and that chCPS bound IMP, although none of these interactions led to changes in enzymatic activity. These findings strongly suggest that the nonresponsive sCPS is not able to communicate occupancy of the allosteric site to the active site but does contain a latent allosteric interaction domain.Carbamoyl phosphate (CP) 1 is a high energy phosphate compound that plays a key role in the introduction of both ammonia and single carbon units into the metabolic pool. CP formation is catalyzed by carbamoyl-phosphate synthetase (CPS) and the labile metabolite is subsequently utilized in two distinct biosynthetic pathways, one producing pyrimidine nucleotides and the other producing arginine and/or urea (1). Feedback inhibition and feed forward activation serve as major control features for almost all CPSs (2). Elucidating the structural basis for this allosteric regulation and for its varying specificity to fit the different physiological roles of CPSs is critical for understanding the overall structure/function relationship for CPSs and for potentially altering their activity in pathological conditions.Escherichia coli and other enteric bacteria have a single enzyme that provides CP for both arginine and pyrimidine biosynthesis. E. coli CPS (eCPS) is allosterically regulated by metabolites from both pathways. Pyrimidine nucleotides, with UMP the most effective, serve as feedback inhibitors for eCPS (3, 4). Because coordinated synthesis of purine and pyrimidine nucleotides is necessary for nucleic acid synthesis, purine nucleotides, with IMP the most effective, serve as allosteric activators for eCPS (3, 4). Ornithine, the co-substrate with CP for the subsequent arginine pathway enzyme ornithine transcarbamoylase, is also an allosteric activator for eCPS (4, 5).The arginine-specific CPSs from Saccharomyces cerevisiae, Bacillus subtilis, and Neurospora crassa are unique in their apparent total lack of allosteric response (6 -8). The regulation of the S. cerevisiae CP...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Screening Of Potential Allosteric Effectors With Chcps-mentioning

confidence: 99%

Section: Screening Of Potential Allosteric Effectors With Chcps-mentioning

confidence: 99%

See 1 more Smart Citation

Unmasking a Functional Allosteric Domain in an Allosterically Nonresponsive Carbamoyl-phosphate Synthetase

Eroglu

Powers‐Lee

2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Previous studies performed with analogs of UMP and IMP already indicated that these antagonistic nucleotide effectors may bind the same or overlapping sites, but that this site is distinct from the ornithine binding site (Boettcher and Meister 1981, 1982; Mora et al 1999; Fresquet et al 2000; Braxton et al 1999). The binding of UMP and ornithine essentially affect the binding of the second Mg 2+ ATP molecule (Braxton et al 1992, 1999).…”

Section: Allosteric Regulation Of Cpsase Activitymentioning

confidence: 99%

Regulation of carbamoylphosphate synthesis in Escherichia coli: an amazing metabolite at the crossroad of arginine and pyrimidine biosynthesis

Charlier

Minh

Roovers³

2018

Amino Acids

View full text Add to dashboard Cite

In all organisms, carbamoylphosphate (CP) is a precursor common to the synthesis of arginine and pyrimidines. In Escherichia coli and most other Gram-negative bacteria, CP is produced by a single enzyme, carbamoylphosphate synthase (CPSase), encoded by the carAB operon. This particular situation poses a question of basic physiological interest: what are the metabolic controls coordinating the synthesis and distribution of this high-energy substance in view of the needs of both pathways? The study of the mechanisms has revealed unexpected moonlighting gene regulatory activities of enzymes and functional links between mechanisms as diverse as gene regulation and site-specific DNA recombination. At the level of enzyme production, various regulatory mechanisms were found to cooperate in a particularly intricate transcriptional control of a pair of tandem promoters. Transcription initiation is modulated by an interplay of several allosteric DNA-binding transcription factors using effector molecules from three different pathways (arginine, pyrimidines, purines), nucleoid-associated factors (NAPs), trigger enzymes (enzymes with a second unlinked gene regulatory function), DNA remodeling (bending and wrapping), UTP-dependent reiterative transcription initiation, and stringent control by the alarmone ppGpp. At the enzyme level, CPSase activity is tightly controlled by allosteric effectors originating from different pathways: an inhibitor (UMP) and two activators (ornithine and IMP) that antagonize the inhibitory effect of UMP. Furthermore, it is worth noticing that all reaction intermediates in the production of CP are extremely reactive and unstable, and protected by tunneling through a 96 Å long internal channel.

show abstract

“…The isolated domains form homodimers that catalyze both ATP-dependent partial reactions and the overall synthesis of carbamoyl phosphate from bicarbonate, NH 3 , and two ATP molecules. The only apparent functional difference between the CPS.A and CPS.B dimers is that the latter is subject to allosteric control because effectors bind to the regulatory subdomain (21)(22)(23)(24)(25)(26)(27) at the extreme carboxyl end of CPS.B.…”

mentioning

confidence: 99%

A Novel Carbamoyl-Phosphate Synthetase from Aquifex aeolicus

Ahuja

Purcarea

Guy

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Aquifex aeolicus, an extreme hyperthermophile, has neither a full-length carbamoyl-phosphate synthetase (CPSase) resembling the enzyme found in all mesophilic organisms nor a carbamate kinase-like CPSase such as those present in several hyperthermophilic archaea. However, the genome has open reading frames encoding putative proteins that are homologous to the major CPSase domains. The glutaminase, CPS.A, and CPS.B homologs from A. aeolicus were cloned, overexpressed in Escherichia coli, and purified to homogeneity. The isolated proteins could catalyze several partial reactions but not the overall synthesis of carbamoyl phosphate. However, a stable 124-kDa complex could be reconstituted from stoichiometric amounts of CPS.A and CPS.B proteins that synthesized carbamoyl phosphate from ATP, bicarbonate, and ammonia. The inclusion of the glutaminase subunit resulted in the formation of a 171-kDa complex that could utilize glutamine as the nitrogen-donating substrate, although the catalytic efficiency was significantly compromised. Molecular modeling, using E. coli CPSase as a template, showed that the enzyme has a similar structural organization and interdomain interfaces and that all of the residues known to be essential for function are conserved and properly positioned. A steady state kinetic study at 78°C indicated that although the substrate affinity was similar for bicarbonate, ammonia, and glutamine, the K m for ATP was appreciably higher than that of any known CPSase. The A. aeolicus complex, with a split gene encoding the major synthetase domains and relatively inefficient coupling of amidotransferase and synthetase functions, may be more closely related to the ancestral precursor of contemporary mesophilic CPSases.Carbamoyl phosphate is the initial intermediate in the biosynthesis of both pyrimidine and arginine in all organisms and of urea in ureotelic species. In most mesophiles, carbamoylphosphate synthetase (CPSase) 1 (EC 6.3.5.5) catalyzes the following reaction.The enzyme from Escherichia coli consists of a 120-kDa synthetase subunit (CPS) and a 40-kDa amidotransferase or glutaminase subunit (GLN) (1). Upon dissociation of the heterodimer (2, 3), the GLN subunit was found to catalyze the hydrolysis of glutamine, whereas the CPS subunit catalyzed the synthesis of carbamoyl phosphate from ammonia, bicarbonate, and ATP. The GLN and CPS domains are fused in CAD (4 -6), a mammalian multifunctional protein that catalyzes the first three steps of the de novo pyrimidine biosynthetic pathway. Although glutamine hydrolysis is the usual source of ammonia for carbamoyl phosphate synthesis, mitochondrial CPSase I, the enzyme that catalyzes the first step in the urea cycle (1), has an inactive homolog of the GLN subunit fused to the amino end of the synthetase subunit. Consequently, CPSase I cannot hydrolyze glutamine, and instead it uses ammonia directly as the nitrogen-donating substrate.Although the CPSases have a diverse structural organization, they share a common catalytic mechanism (1) that proceeds through ...

show abstract

Localization of the site for the nucleotide effectors ofEscherichia colicarbamoyl phosphate synthetase using site‐directed mutagenesis

Cited by 16 publications

References 25 publications

Unmasking a Functional Allosteric Domain in an Allosterically Nonresponsive Carbamoyl-phosphate Synthetase

Unmasking a Functional Allosteric Domain in an Allosterically Nonresponsive Carbamoyl-phosphate Synthetase

Regulation of carbamoylphosphate synthesis in Escherichia coli: an amazing metabolite at the crossroad of arginine and pyrimidine biosynthesis

A Novel Carbamoyl-Phosphate Synthetase from Aquifex aeolicus

Contact Info

Product

Resources

About