Related Experiment Video

Updated: May 21, 2026

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
11:27

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

Published on: May 13, 2020

Function analysis of conserved amino acid residues in a Mn(2+)-dependent protein phosphatase, Pph3, from Myxococcus

Yumi Mori1, Kaoru Takegawa, Yoshio Kimura

  • 1Department of Applied Biological Science, Kagawa University, Miki-cho, Kagawa, Japan.

Journal of Biochemistry
|June 7, 2012
PubMed

Insights

Myxococcus xanthus protein phosphatase Pph3 activity is regulated by its C-terminal region and cyclic nucleotide monophosphate (cNMP) binding. Disulfide bond disruption and cAMP binding influence Pph3

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Myxococcus xanthus protein phosphatase Pph3 is a member of the Mg(2+)- or Mn(2+)-dependent protein phosphatase (PPM) family.
  • Bacterial PPMs are characterized by three divalent metal ions and a flap subdomain, crucial for catalytic activity.
  • Pph3 possesses a cyclic nucleotide monophosphate (cNMP)-binding domain in its C-terminal region and requires 2-mercaptoethanol for activity.

Purpose of the Study:

  • To investigate the role of the C-terminal region and the cNMP-binding domain in the enzymatic activity of Pph3.
  • To elucidate the influence of disulfide bonds and cAMP on Pph3 phosphatase activity.
  • To understand the regulatory mechanisms governing Pph3 function in Myxococcus xanthus.

Main Methods:

  • Site-specific mutations were introduced in putative metal- or phosphate-ion binding sites to assess their impact on enzymatic activity.
  • A C-terminal deletion mutant of Pph3 was constructed and analyzed.
  • Mutations were introduced to disrupt a disulfide bond within the catalytic domain.
  • Enzyme activity was measured in the presence and absence of cAMP and 2-mercaptoethanol.

Main Results:

  • Mutations in metal- or phosphate-ion binding sites significantly reduced Pph3 activity.
  • The C-terminal deletion mutant exhibited high activity without 2-mercaptoethanol, unlike the wild-type enzyme.
  • Wild-type Pph3 activity was enhanced by cAMP, while a triple mutant of the cNMP-binding domain showed reduced activity.
  • Disruption of the disulfide bond increased activity in the absence of 2-mercaptoethanol, particularly in the wild-type enzyme.

Conclusions:

  • The C-terminal region of Pph3 likely influences the formation of a disulfide bond in the catalytic domain.
  • Cleavage of this disulfide bond by 2-mercaptoethanol is facilitated by cAMP binding.
  • These findings suggest a complex regulatory mechanism for Pph3 involving its C-terminal region, disulfide bonds, and cNMP binding.

More Related Videos

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors10:17

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Published on: April 29, 2022

Identification of Post-translational Modifications of Plant Protein Complexes10:07

Identification of Post-translational Modifications of Plant Protein Complexes

Published on: February 22, 2014

Related Concept Videos

Protein Kinases and Phosphatases02:54

Protein Kinases and Phosphatases

Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
Conserved Binding Sites01:49

Conserved Binding Sites

Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally analyses the...
Phosphorylation01:02

Phosphorylation

The addition or removal of phosphate groups from proteins is the most common chemical modification that regulates cellular processes. These modifications can affect the structure, activity, stability, and localization of proteins within cells as well as their interactions with other proteins.
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
Conservation of Protein Domains Over Different Proteins02:26

Conservation of Protein Domains Over Different Proteins

Protein domains are small structurally independent units that are part of a single amino acid chain.  Although these domains are often structurally independent, they may rely on synergistic effects to perform their functions as part of a larger protein. Protein domains may be conserved within the same organism, as well as across different organisms.
A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to form...
Covalently Linked Protein Regulators02:04

Covalently Linked Protein Regulators

Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein.