Microbial proteases constitute one of the most important groups of industrially relevant enzymes. Proline iminopeptidases (PIPs) that specifically release amino-terminal proline from peptides are of major interest for applications in food biotechnology. Proline iminopeptidase has been extensively characterised in bacteria and filamentous fungi. However, no similar reports exist for yeasts. In this study, a protease gene from Glaciozyma antarctica designated as GaPIP was cloned and overexpressed in Escherichia coli. Sequence analyses of the gene revealed a 960 bp open reading frame encoding a 319 amino acid protein (35,406 Da). The purified recombinant GaPIP showed a specific activity of 3561 Umg−1 towards L-proline-p-nitroanilide, confirming its identity as a proline iminopeptidase. GaPIP is a cold-active enzyme with an optimum activity of 30 °C at pH 7.0. The enzyme is stable between pH 7.0 and 8.0 and able to retain its activity at 10–30 °C. Although GaPIP is a serine protease, only 25% inhibition by the serine protease inhibitor, phenylmethanesulfonylfluoride (PMSF) was recorded. This enzyme is strongly inhibited by the presence of EDTA, suggesting that it is a metalloenzyme. The dimeric structure of GaPIP was determined at a resolution of 2.4 Å. To date, GaPIP is the first characterised PIP from yeasts and the structure of GaPIP is the first structure for PIP from eukaryotes.
Abstract PSD-95 is a member of Membrane Associated Guanylate Kinase class of proteins which form scaffolding interactions with partner proteins including ion and receptor channels. PSD-95 is directly implicated in modulating the electrical responses of excitable cells. The first two PSD-95/Disks Large/Zona Occludens domains of PSD-95 have been shown to be the key component in the formation of channel clusters. We report crystal structures of the dual domain in both in apo and ligand-bound form; thermodynamic analysis of ligand association and Small Angle X-ray Scattering of the dual domain in the absence and presence of ligands. These experiments reveal that the ligated double domain forms a scaffold in the complete sense of the word. The concentration of the components in this study is comparable to those found in compartments of excitable cells such as the postsynaptic density and juxta-paranodes of Ranvier. The properties of the dual domain explain the basis of the scaffolding function of PSD-95, and provide a more detailed understanding of the integration of key components of neuronal specializations involved in nervous signal transmission.
PSD-95 is a member of the membrane-associated guanylate kinase class of proteins that forms scaffolding interactions with partner proteins, including ion and receptor channels. PSD-95 is directly implicated in modulating the electrical responses of excitable cells. The first two PSD-95/disks large/zona occludens (PDZ) domains of PSD-95 have been shown to be the key component in the formation of channel clusters. We report crystal structures of this dual domain in both apo- and ligand-bound form: thermodynamic analysis of the ligand association and small-angle x-ray scattering of the dual domain in the absence and presence of ligands. These experiments reveal that the ligated double domain forms a three-dimensional scaffold that can be described by a space group. The concentration of the components in this study is comparable with those found in compartments of excitable cells such as the postsynaptic density and juxtaparanodes of Ranvier. These in vitro experiments inform the basis of the scaffolding function of PSD-95 and provide a detailed model for scaffold formation by the PDZ domains of PSD-95.
Aims:To date, nine classes of copper regulation mechanisms have been discovered in bacteria and CsoR regulator protein is the most recent among them.Only a few have been structurally and functionally characterized.The present study was aimed to isolate and characterize the sequence, structure and biochemical properties of CsoRGz-like hypothetical protein (HP) to be potentially used as a copper sensor protein. Methodology and results:A scan of the complete genome of a Geobacillus zalihae strain T1 revealed the presence of CsoR-like (CsoRGz) HP, which contains CsoR-like_DUF156 domain and highly conserved Cys-His-Cys residues essential for copper binding.It only shares moderate sequence identity with structurally characterized CsoR proteins.CsoRGz-like HP was subjected to sequence analyses to identify important domains, motifs and residues, while circular dichroism and X-ray crystallography were used to determine its secondary and tertiary structures.CsoRGz appears to be a dimer comprising mainly α-helices, with putative, conserved metal-binding ligands, Cys46-His71-Cys75, located on the α2 helix of the protein.Biophysical characterization of CsoRGz using UV/Vis and fluorescence spectrophotometry confirmed its interaction with Cu(I).Docking of Cu(I) to the dimeric structure of CsoRGz showed that Cu(I) could be coordinated by the above metal-binding residues, further stabilized by the hydrophobic core at the metal-binding site. Conclusion, significance and impact of study:The findings in this study suggest that CsoRGz-like HP might be a novel CsoR protein.This adds to the breadth and numbers of possible CsoR proteins, particularly uncharacterized ones, existing in the pool of hypothetical proteins, to be further characterized and compared across bacterial taxa.
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