Dissection of the Conduit for Allosteric Control of Carbamoyl Phosphate Synthetase by Ornithine

Pierrat, Olivier A.; Javid-Majd, Farah; Raushel, Frank M.

doi:10.1006/abbi.2002.2768

Cited by 7 publications

(10 citation statements)

References 26 publications

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“…Ornithine binding has been localized to the interface of the C/D domains by crystal structure analysis (29,30) and site-directed mutagenesis (35,36,42,43). Domain D also appears to be the allosteric domain for the other CPSs that have been studied thus far (44,45).…”

Section: Resultsmentioning

confidence: 99%

“…The ␦-amino group of ornithine interacts with domain C, forming hydrogen bonds with O ⑀1 of Glu-783, O of Asp-791, and O ⑀1 of Glu-892. In confirmation of these proposed roles, site-directed mutation of Glu-783, Glu-892, and Thr-1042 yielded greatly diminished binding of ornithine without significantly affecting UMP/IMP binding (42,43).…”

Section: Screening Of Potential Allosteric Effectors With Chcps-mentioning

confidence: 89%

“…The addition of UMP caused a 30% decrease in chCPS activity versus a 72% decrease for eCPS. 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).…”

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%

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

Eroglu

Powers‐Lee

2002

Journal of Biological Chemistry

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Screening Of Potential Allosteric Effectors With Chcps-mentioning

confidence: 89%

Section: Screening Of Potential Allosteric Effectors With Chcps-mentioning

confidence: 99%

Section: Screening Of Potential Allosteric Effectors With Chcps-mentioning

confidence: 99%

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

Eroglu

Powers‐Lee

2002

Journal of Biological Chemistry

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“…Sequence determination and biochemical characterization of carB mutants isolated by Mergeay et al (1974) pinpointed the crucial role of this residue in the allosteric regulation of CPSase activity (Delannay et al 1999). Substitution of Thr1042 by Ileu greatly lowers the capacity of ornithine activation, but the enzyme is still sensitive to UMP and IMP, although to a lower extent (Delannay et al 1999; Rochera et al 2002; Pierrat et al 2002). Similarly, substitution of Ser948 by Phe results in an enzyme that is insensitive to UMP and IMP, but still activated by ornithine, though again to a reduced extent (Delannay et al 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

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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.

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A Functional Analysis of the Allosteric Nucleotide Monophosphate Binding Site of Carbamoyl Phosphate Synthetase

Pierrat

Raushel

2002

Archives of Biochemistry and Biophysics

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Dissection of the Conduit for Allosteric Control of Carbamoyl Phosphate Synthetase by Ornithine

Cited by 7 publications

References 26 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 Functional Analysis of the Allosteric Nucleotide Monophosphate Binding Site of Carbamoyl Phosphate Synthetase

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