Differential roles for three conserved histidine residues within the large subunit of carbamoyl phosphate synthetase

Biochemistry ◽  
1993 ◽  
Vol 32 (1) ◽  
pp. 232-240 ◽  
Author(s):  
Bryant W. Miles ◽  
Shadreck M. Mareya ◽  
Laura E. Post ◽  
David J. Post ◽  
Sun Hee Chang ◽  
...  
Keyword(s):  
Large Subunit ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Histidine Residues

Site-directed mutagenesis studies on the uridine monophosphate binding sites of feedback inhibition in carbamoyl phosphate synthetase and effects on cytidine production by Bacillus amyloliquefaciens

2013 ◽  
Vol 59 (6) ◽  
pp. 374-379 ◽  
Author(s):  
Haitian Fang ◽  
Huiyan Liu ◽  
Ning Chen ◽  
Chenglin Zhang ◽  
Xixian Xie ◽  
...  
Keyword(s):  
Binding Sites ◽  
Bacillus Amyloliquefaciens ◽  
De Novo ◽  
Feedback Inhibition ◽  
Large Subunit ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Uridine Monophosphate

A major problem when pyrimidine de novo biosynthesis is used for cytidine production is the existence of many negative regulatory factors. Cytidine biosynthesis in Bacillus amyloliquefaciens proceeds via a pathway that is controlled by uridine monophosphate (UMP) through feedback inhibition of carbamoyl phosphate synthetase (CPS), the enzyme that converts CO2, NH3, and glutamine to carbamoyl phosphate. In this study, the gene carB encoding the large subunit of CPS from B. amyloliquefaciens CYT1 was site directed, and the UMP binding sites of feedback inhibition in Bam-CPS are described. The residues Thr-941, Thr-970, and Lys-986 in CPS from B. amyloliquefaciens were subjected to site-directed mutagenesis to alter UMP’s feedback inhibition of CPS. To find feedback-resistant B. amyloliquefaciens, the influence of the T941F, T970A, K986I, T941F/K986I, and T941F/T970A/K986I mutations on CPS enzymatic properties was studied. The recombinant B. amyloliquefaciens with mutated T941F/K986I and T941F/T970A/K986I CPS showed a 3.7- and 5.7-fold increase, respectively, in cytidine production in comparison with the control expressing wild-type CPS, which was more suitable for further application of the cytidine synthesis. To a certain extent, the 5 mutations were found to release the enzyme from UMP inhibition and to improve B. amyloliquefaciens cytidine-producing strains.

scholarly journals Specific Discrimination of Three Pathogenic Salmonella enterica subsp. enterica Serotypes bycarB-Based Oligonucleotide Microarray

2013 ◽  
Vol 80 (1) ◽  
pp. 366-373 ◽  
Author(s):  
Hwa Hui Shin ◽  
Byeong Hee Hwang ◽  
Jeong Hyun Seo ◽  
Hyung Joon Cha
Keyword(s):  
Salmonella Enterica ◽  
Pathogenic Bacteria ◽  
Detection System ◽  
Large Subunit ◽  
Cross Reactivity ◽  
Oligonucleotide Microarray ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Content Type ◽  
Contaminated Food

ABSTRACTIt is important to rapidly and selectively detect and analyze pathogenicSalmonella entericasubsp.entericain contaminated food to reduce the morbidity and mortality ofSalmonellainfection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on thecarBgene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate threeS. entericasubsp.entericaserotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the targetSalmonellaserotype from a bacterial mixture. Therefore, these results demonstrated that our novelcarB-based oligonucleotide microarray can be used as an effective and specific detection system forS. entericasubsp.entericaserotypes.

Molecular studies on an ancient gene encoding for carbamoyl-phosphate synthetase

1993 ◽  
Vol 84 (2) ◽  
pp. 119-128 ◽  
Author(s):  
J. P. Schofield
Keyword(s):  
Large Subunit ◽  
Methanosarcina Barkeri ◽  
Cosmid Library ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Gene Encoding ◽  
Ancestral Gene ◽  
Carbamoylphosphate Synthetase ◽  
Old Evidence ◽  
I Gene

1. Carbamoyl-phosphate synthetase (EC 6.3.5.5.) catalyses the synthesis of carbamoyl phosphate, the immediate precursor of arginine and pyrimidine biosynthesis, and is highly conserved throughout evolution. The large subunit of all carbamoyl-phosphate synthetases sequenced to date comprises two highly homologous halves, the product of a proposed ancestral gene duplication. The sequences of the enzymes of Escherichia coli, Drosophila melanogaster, Saccharomyces cerevisiae, rat and Syrian hamster all have duplications, suggesting that this event occurred in the progenote predating the separation of the major phylae. Until now, only limited data on carbamoyl-phosphate synthetase were available for the primitive eukaryote Dictyostelium discoideum and for the Archaea Methanosarcina barkeri MS. The DNA sequence of the D. discoideum carbamoylphosphate gene and additional sequence for the carbamoyl-phosphate synthetase gene of M. barkeri MS have been determined, and a duplicated structure for both is clearly demonstrated. 2. Genes with ancient duplications provide unique information on their evolution. A study of the intron/exon organization of the rat carbamoylphosphate synthetase I gene and the carbamoylphosphate synthetase hamster II gene in the CAD multi-gene complex shows that at least some of their introns are very old. Evidence is provided that some introns must have been present in the ancestral precursor before its duplication. 3. The human carbamoyl-phosphate synthetase I gene has been isolated and characterized. A human liver cDNA library was constructed and probed for carbamoyl-phosphate synthetase I. A human genomic DNA cosmid library was also probed for the carbamoyl-phosphate synthetase I gene. The cDNA sequence of the human carbamoyl-phosphate synthetase I gene has been determined, and work has been initiated to confirm that at least part of this gene is contained within two cosmids spanning 46 kb. This will enable future studies to be made on mutations in this gene in the rare autosomal recessive deficiency of carbamoyl-phosphate synthetase I.

Role of the four conserved histidine residues in the amidotransferase domain of carbamoyl phosphate synthetase

Biochemistry ◽  
1991 ◽  
Vol 30 (32) ◽  
pp. 7901-7907 ◽  
Author(s):  
Sophie Gaillard Miran ◽  
Sun Hee Chang ◽  
Frank M. Raushel
Keyword(s):  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Histidine Residues

scholarly journals A Genetic Determinant of Persister Cell Formation in Bacterial Pathogens

2018 ◽  
Vol 200 (17) ◽  
Author(s):  
David R. Cameron ◽  
Yue Shan ◽  
Eliza A. Zalis ◽  
Vincent Isabella ◽  
Kim Lewis
Keyword(s):  
Cystic Fibrosis ◽  
Antibiotic Treatment ◽  
Bacterial Pathogens ◽  
Large Subunit ◽  
General Mechanism ◽  
Antibiotic Tolerance ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Content Type ◽  
Intracellular Atp

ABSTRACTPersisters represent a small subpopulation of cells within a bacterial culture that are tolerant to killing by antibiotics. Persisters have been linked to recalcitrant infections caused by numerous bacterial pathogens, includingPseudomonas aeruginosa. A classic example is the incurable infection of the airways for patients with cystic fibrosis. The genetic mediators of persister formation forP. aeruginosaare poorly understood. We generated a high-density transposon insertion library ofP. aeruginosaPAO1 and determined the relative frequency of each insertion following fluoroquinolone treatment using transposon sequencing (Tn-seq). Of the 4,411 disrupted genes included in the screen, 137 had a ≥10-fold impact on survival. The gene disruption that resulted in the lowest survival rate was disruption ofcarB, which codes for the large subunit of carbamoyl phosphate synthetase (CPSase). CPSase is a metabolic enzyme that is involved in pyrimidine and arginine synthesis. Disruption ofcarBresulted in survival rates that were reduced by up to 2,500-fold following antibiotic treatment, and this phenotype was abolished by the addition of uracil, highlighting the importance ofde novopyrimidine biosynthesis for persister formation. Disruption ofcarBresulted in intracellular ATP accumulation, and lowering ATP levels using arsenate restored the antibiotic tolerance profile of the mutant to levels similar to those seen with the wild type. A decrease in ATP would lead to reduced antibiotic target activity and increased survival.IMPORTANCEAntibiotic treatment ofP. aeruginosaresiding in the lung of cystic fibrosis patients is ineffective. Treatment failure is attributed in part to antibiotic-tolerant phenotypic variants known as persister cells. Understanding how these cells emerge will likely inform future therapeutic strategies. In the current study, we identifiedcarB, which codes for the large subunit of carbamoyl-phosphate synthetase, as a persister gene that contributes to multidrug tolerance inP. aeruginosa. Disruption ofcarBresulted in a metabolic perturbation that increased cellular ATP and reduced persister formation. Conversely, lowering ATP in the mutant restored antibiotic tolerance. Our data support the hypothesis that a drop in intracellular ATP is a general mechanism of persister formation in bacteria.

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