scholarly journals Tautomeric state and pKa of the phosphorylated active site histidine in the N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: Sugar phosphotransferase system

1998 ◽  
Vol 7 (3) ◽  
pp. 789-793 ◽  
Author(s):  
Daniel S. Garrett ◽  
G.Marius Clore ◽  
Angela M. Gronenborn ◽  
Yeong-Jae Seok ◽  
Alan Peterkfsky
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Phosphotransferase System ◽  
Terminal Domain ◽  
Enzyme I ◽  
Active Site Histidine

Properties of phosphorylated protein intermediates of the bacterial phosphoenolpyruvate:sugar phosphotransferase system

1992 ◽  
Vol 70 (3-4) ◽  
pp. 242-246 ◽  
Author(s):  
J. W. Anderson ◽  
E. B. Waygood ◽  
M. H. Saier Jr. ◽  
J. Reizer
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Active Site ◽  
Active Sites ◽  
Sugar Transport ◽  
Wild Type ◽  
Phosphotransferase System ◽  
E Coli ◽  
Phosphorylated Protein ◽  
Enzyme I

The phosphohydrolysis properties of the following phosphoprotein intermediates of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) were investigated: enzyme I, HPr, and the IIAGlc domain of the glucose enzyme II of Bacillus subtilis; and IIAGlc (fast and slow forms) of Escherichia coli. The phosphohydrolysis properties were also studied for the site-directed mutant H68A of B. subtilis IIAGlc. Several conclusions were reached. (i) The phosphohydrolysis properties of the homologous phosphoprotein intermediates of B. subtilis and E. coli are similar. (ii) These properties deviate from those of isolated Nδ1- and Nε2-phosphohistidine indicating the participation of neighbouring residues at the active sites of these proteins. (iii) The rates of phosphohydrolysis of the H68A mutant of B. subtilis IIAGlc were reduced compared with the wild-type protein, suggesting that both His-83 and His-68 are present at the active site of wild-type IIAGlc. (iv) The removal of seven N-terminal residues of E. coli IIAGlc reduced the rates of phosphohydrolysis between pH 5 and 8.Key words: phosphoenolpyruvate:sugar phosphotransferase system, phosphoproteins, phosphohistidine, phosphorylation, sugar transport.

scholarly journals Mutational Inactivation of a Gene Homologous to Escherichia coli ptsP Affects Poly-β-Hydroxybutyrate Accumulation and Nitrogen Fixation in Azotobacter vinelandii

1998 ◽  
Vol 180 (18) ◽  
pp. 4790-4798 ◽  
Author(s):  
Daniel Segura ◽  
Guadalupe Espín
Keyword(s):  
Escherichia Coli ◽  
Azotobacter Vinelandii ◽  
Growth Defect ◽  
Nucleotide Sequencing ◽  
Acetylene Reduction Activity ◽  
Respiratory Protection ◽  
Phosphotransferase System ◽  
Terminal Domain ◽  
Diazotrophic Growth ◽  
Enzyme I

ABSTRACT Strain DS988, an Azotobacter vinelandii mutant with a reduced capacity to accumulate poly-β-hydroxybutyrate, was isolated after mini-Tn5 mutagenesis of the UW136 strain. Cloning and nucleotide sequencing of the affected locus revealed a gene homologous to Escherichia coli ptsP which encodes enzyme INtr, a homologue of enzyme I of the phosphoenol pyruvate-sugar phosphotransferase system with an N-terminal domain similar to the N-terminal domain of some NifA proteins. Strain DS988 was unable to grow diazotrophically with 10 mM glucose as a carbon source. Diazotrophic growth on alternative carbon sources such as gluconate was only slightly affected. Glucose uptake, as well as glucose kinase and glucose-6-phosphate-dehydrogenase activities that lead to the synthesis of gluconate-6-phosphate, were not affected by the ptsP mutation. The inability of DS988 to grow diazotrophically in 10 mM glucose was overcome by supplying ammonium or other sources of fixed nitrogen. Acetylene reduction activity but not transcription of the nitrogenase structural gene nifH was shown to be impaired in strain DS988 when it was incubated in 10 mM glucose. The diazotrophic growth defect of DS988 was restored either by increasing the glucose concentration to above 20 mM or by lowering the oxygen concentration. These data suggest that a mutation inptsP leads to a failure in poly-β-hydroxybutyrate metabolism and in the respiratory protection of nitrogenase under carbon-limiting conditions.

scholarly journals A Single Point Mutation Controls the Rate of Interconversion Between the g+ and g− Rotamers of the Histidine 189 χ2 Angle That Activates Bacterial Enzyme I for Catalysis

2021 ◽  
Vol 8 ◽  
Author(s):  
Jeffrey A. Purslow ◽  
Jolene N. Thimmesch ◽  
Valeria Sivo ◽  
Trang T. Nguyen ◽  
Balabhadra Khatiwada ◽  
...  
Keyword(s):  
Protein Design ◽  
Active Site ◽  
Single Point ◽  
Conformational Dynamics ◽  
Phosphorylation Site ◽  
Phosphoryl Group ◽  
Single Point Mutation ◽  
Phosphotransferase System ◽  
Function Relationship ◽  
Enzyme I

Enzyme I (EI) of the bacterial phosphotransferase system (PTS) is a master regulator of bacterial metabolism and a promising target for development of a new class of broad-spectrum antibiotics. The catalytic activity of EI is mediated by several intradomain, interdomain, and intersubunit conformational equilibria. Therefore, in addition to its relevance as a drug target, EI is also a good model for investigating the dynamics/function relationship in multidomain, oligomeric proteins. Here, we use solution NMR and protein design to investigate how the conformational dynamics occurring within the N-terminal domain (EIN) affect the activity of EI. We show that the rotameric g+-to-g− transition of the active site residue His189 χ2 angle is decoupled from the state A-to-state B transition that describes a ∼90° rigid-body rearrangement of the EIN subdomains upon transition of the full-length enzyme to its catalytically competent closed form. In addition, we engineered EIN constructs with modulated conformational dynamics by hybridizing EIN from mesophilic and thermophilic species, and used these chimeras to assess the effect of increased or decreased active site flexibility on the enzymatic activity of EI. Our results indicate that the rate of the autophosphorylation reaction catalyzed by EI is independent from the kinetics of the g+-to-g− rotameric transition that exposes the phosphorylation site on EIN to the incoming phosphoryl group. In addition, our work provides an example of how engineering of hybrid mesophilic/thermophilic chimeras can assist investigations of the dynamics/function relationship in proteins, therefore opening new possibilities in biophysics.

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