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.

scholarly journals Structural insight into glucose repression of the mannitol operon

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mangyu Choe ◽  
Huitae Min ◽  
Young-Ha Park ◽  
Yeon-Ran Kim ◽  
Jae-Sung Woo ◽  
...  
Keyword(s):  
Glucose Repression ◽  
Phosphorylation Site ◽  
Phosphotransferase System ◽  
E Coli ◽  
Carbon Compounds ◽  
Structural Insight ◽  
Enzyme I ◽  
Insight Into ◽  
Repressor Activity

Abstract Carbon catabolite repression is a regulatory mechanism to ensure sequential utilization of carbohydrates and is usually accomplished by repression of genes for the transport and metabolism of less preferred carbon compounds by a more preferred one. Although glucose and mannitol share the general components, enzyme I and HPr, of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) for their transport, glucose represses the transport and metabolism of mannitol in a manner dependent on the mannitol operon repressor MtlR in Escherichia coli. In a recent study, we identified the dephosphorylated form of HPr as a regulator determining the glucose preference over mannitol by interacting with and augmenting the repressor activity of MtlR in E. coli. Here, we determined the X-ray structure of the MtlR-HPr complex at 3.5 Å resolution to understand how phosphorylation of HPr impedes its interaction with MtlR. The phosphorylation site (His15) of HPr is located close to Glu108 and Glu140 of MtlR and phosphorylation at His15 causes electrostatic repulsion between the two proteins. Based on this structural insight and comparative sequence analyses, we suggest that the determination of the glucose preference over mannitol solely by the MtlR-HPr interaction is conserved within  the Enterobacteriaceae family.

scholarly journals Design to Data for mutants of β-glucosidase B from Paenibacillus polymyxa: L171M, H178M, M221L, E406W, N160E, F415M

2020 ◽  
Author(s):  
Xiaoqing Huang ◽  
Daniel Kim ◽  
Peishan Huang ◽  
Ashley Vater ◽  
Justin B. Siegel
Keyword(s):  
Thermal Stability ◽  
Protein Design ◽  
Single Point ◽  
Point Mutations ◽  
Paenibacillus Polymyxa ◽  
Biophysical Parameters ◽  
Limited Degree ◽  
Function Relationship ◽  
The Stability ◽  
And Function

ABSTRACTComputational protein design is growing in popularity as a means to engineer enzymes. Currently, protein design algorithms can predict the stability and function of the enzymes to only a limited degree. Thus, further experimental data is required for training software to more accurately characterize the structure-function relationship of enzymes. To date, the Design2Data (D2D) database holds 129 single point mutations of β-glucosidase B (BglB) characterized by kinetic and thermal stability biophysical parameters. In this study, we introduced six mutants into the BglB database and examined their catalytic activity and thermal stability: L171M, H178M, M221L, E406W, N160E, and F415M.

Structures and homologies of carbohydrate: phosphotransferase system (PTS) proteins

1990 ◽  
Vol 326 (1236) ◽  
pp. 489-504 ◽  
Keyword(s):  
Regulatory Network ◽  
Phosphorylation Site ◽  
Transport Systems ◽  
Gram Negative Bacteria ◽  
Structural Genes ◽  
Phosphotransferase System ◽  
Membrane Bound ◽  
Bacterial Phosphotransferase System ◽  
Group Translocation ◽  
Enzyme I

The bacterial phosphotransferase system (PTS) is the major transport system for many carbohydrates that are phosphorylated concomitantly with the translocation step through the membrane (group translocation). It consists of two general proteins, enzyme I and histidine protein (HPr), and a series of more than 15 substrate-specific enzymes II (ElI). The sequences of several of these derived from Gram-positive and Gram-negative bacteria were compared, which allowed the possible identification of the following functional domains: membrane-bound pore, substrate-binding site, linker domains, transphosphorylation domain and primary phosphorylation site. Several Ells have been analysed in the meantime, also by topological .tests, by sequential deletion of the corresponding structural genes, and by construction of intergenic hybrids between different domains of several Ells. These data suggest evolutionary relationships between different Ells; they also enable a general model to be constructed of Ells as carbohydrate transport systems, phosphotransferases, chemoreceptors in chemotaxis and as part of a global regulatory network.

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 Yersinia pestisPla Protein Thwarts T Cell Defense against Plague

2019 ◽  
Vol 87 (5) ◽  
Author(s):  
Stephen T. Smiley ◽  
Frank M. Szaba ◽  
Lawrence W. Kummer ◽  
Debra K. Duso ◽  
Jr-Shiuan Lin
Keyword(s):  
T Cell ◽  
Active Site ◽  
Single Point ◽  
Single Point Mutation ◽  
Human Pathogens ◽  
Mechanistic Studies ◽  
Therapeutic Approaches ◽  
Content Type ◽  
Cell Defense ◽  
Cell Responses

ABSTRACTPlague is a rapidly lethal human disease caused by the bacteriumYersinia pestis. This study demonstrated that theY. pestisplasminogen activator Pla, a protease that promotes fibrin degradation, thwarts T cell-mediated defense against fully virulentY. pestis. Introducing a single point mutation into the active site of Pla suffices to render fully virulentY. pestissusceptible to primed T cells. Mechanistic studies revealed essential roles for fibrin during T cell-mediated defense against Pla-mutantY. pestis. Moreover, the efficacy of T cell-mediated protection against variousY. pestisstrains displayed an inverse relationship with their levels of Pla activity. Together, these data indicate that Pla functions to thwart fibrin-dependent T cell-mediated defense against plague. Other important human bacterial pathogens, including staphylococci, streptococci, and borrelia, likewise produce virulence factors that promote fibrin degradation. The discovery thatY. pestisthwarts T cell defense by promoting fibrinolysis suggests novel therapeutic approaches to amplifying T cell responses against human pathogens.

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