Structural and biochemical characterization of the Clostridium perfringens-specific Zn2+-dependent amidase endolysin, Psa, catalytic domain

2021 ◽  
Author(s):  
Hiroshi Sekiya ◽  
Shigehiro Kamitori ◽  
Hirofumi Nariya ◽  
Risa Matsunami ◽  
Eiji Tamai
Keyword(s):  
Clostridium Perfringens ◽  
Biochemical Characterization ◽  
Catalytic Domain

Biochemical Characterization of the Catalytic Domain of Membrane-Type 4 Matrix Metalloproteinase

1999 ◽  
Vol 380 (9) ◽  
Author(s):  
Hansjörg Kolkenbrock ◽  
Lutz Essers ◽  
Norbert Ulbrich ◽  
Horst Will
Keyword(s):  
Matrix Metalloproteinase ◽  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Membrane Type

scholarly journals Thrombin A-Chain: Activation Remnant or Allosteric Effector?

Thrombosis ◽  
2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Isis S. R. Carter ◽  
Amanda L. Vanden Hoek ◽  
Edward L. G. Pryzdial ◽  
Ross T. A. MacGillivray
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Current Data ◽  
Enzymatic Reactions ◽  
Allosteric Effector ◽  
Naturally Occurring ◽  
Physiologic Function ◽  
A Chain

Although prothrombin is one of the most widely studied enzymes in biology, the role of the thrombin A-chain has been neglected in comparison to the other domains. This paper summarizes the current data on the prothrombin catalytic domain A-chain region and the subsequent thrombin A-chain. Attention is given to biochemical characterization of naturally occurring prothrombin A-chain mutations and alanine scanning mutants in this region. While originally considered to be simply an activation remnant with little physiologic function, the thrombin A-chain is now thought to play a role as an allosteric effector in enzymatic reactions and may also be a structural scaffold to stabilize the protease domain.

scholarly journals Characterization of Acp, a Peptidoglycan Hydrolase of Clostridium perfringens with N-Acetylglucosaminidase Activity That Is Implicated in Cell Separation and Stress-Induced Autolysis

2010 ◽  
Vol 192 (9) ◽  
pp. 2373-2384 ◽  
Author(s):  
Emilie Camiade ◽  
Johann Peltier ◽  
Ingrid Bourgeois ◽  
Evelyne Couture-Tosi ◽  
Pascal Courtin ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Clostridium Perfringens ◽  
Vegetative Growth ◽  
Cell Separation ◽  
Catalytic Domain ◽  
Peptidoglycan Hydrolase ◽  
Wild Type ◽  
Induced Autolysis

ABSTRACT This work reports the characterization of the first known peptidoglycan hydrolase (Acp) produced mainly during vegetative growth of Clostridium perfringens. Acp has a modular structure with three domains: a signal peptide domain, an N-terminal domain with repeated sequences, and a C-terminal catalytic domain. The purified recombinant catalytic domain of Acp displayed lytic activity on the cell walls of several Gram-positive bacterial species. Its hydrolytic specificity was established by analyzing the Bacillus subtilis peptidoglycan digestion products by coupling reverse phase-high-pressure liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis, which displayed an N-acetylglucosaminidase activity. The study of acp expression showed a constant expression during growth, which suggested an important role of Acp in growth of C. perfringens. Furthermore, cell fractionation and indirect immunofluorescence staining using anti-Acp antibodies revealed that Acp is located at the septal peptidoglycan of vegetative cells during exponential growth phase, indicating a role in cell separation or division of C. perfringens. A knockout acp mutant strain was obtained by using the insertion of mobile group II intron strategy (ClosTron). The microscopic examination indicated a lack of vegetative cell separation in the acp mutant strain, as well as the wild-type strain incubated with anti-Acp antibodies, demonstrating the critical role of Acp in cell separation. The comparative responses of wild-type and acp mutant strains to stresses induced by Triton X-100, bile salts, and vancomycin revealed an implication of Acp in autolysis induced by these stresses. Overall, Acp appears as a major cell wall N-acetylglucosaminidase implicated in both vegetative growth and stress-induced autolysis.

scholarly journals A recently evolved diflavin-containing monomeric nitrate reductase is responsible for highly efficient bacterial nitrate assimilation

2020 ◽  
Vol 295 (15) ◽  
pp. 5051-5066 ◽  
Author(s):  
Wei Tan ◽  
Tian-Hua Liao ◽  
Jin Wang ◽  
Yu Ye ◽  
Yu-Chen Wei ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Inorganic Nitrogen ◽  
Nitrate Assimilation ◽  
Nitrogen Sources ◽  
Electron Transfer Mechanism ◽  
Cofactor Binding ◽  
Environmental Mycobacteria

Nitrate is one of the major inorganic nitrogen sources for microbes. Many bacterial and archaeal lineages have the capacity to express assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite. Although a nitrate assimilatory pathway in mycobacteria has been proposed and validated physiologically and genetically, the putative NAS enzyme has yet to be identified. Here, we report the characterization of a novel NAS encoded by Mycolicibacterium smegmatis Msmeg_4206, designated NasN, which differs from the canonical NASs in its structure, electron transfer mechanism, enzymatic properties, and phylogenetic distribution. Using sequence analysis and biochemical characterization, we found that NasN is an NADPH-dependent, diflavin-containing monomeric enzyme composed of a canonical molybdopterin cofactor-binding catalytic domain and an FMN–FAD/NAD-binding, electron-receiving/transferring domain, making it unique among all previously reported hetero-oligomeric NASs. Genetic studies revealed that NasN is essential for aerobic M. smegmatis growth on nitrate as the sole nitrogen source and that the global transcriptional regulator GlnR regulates nasN expression. Moreover, unlike the NADH-dependent heterodimeric NAS enzyme, NasN efficiently supports bacterial growth under nitrate-limiting conditions, likely due to its significantly greater catalytic activity and oxygen tolerance. Results from a phylogenetic analysis suggested that the nasN gene is more recently evolved than those encoding other NASs and that its distribution is limited mainly to Actinobacteria and Proteobacteria. We observed that among mycobacterial species, most fast-growing environmental mycobacteria carry nasN, but that it is largely lacking in slow-growing pathogenic mycobacteria because of multiple independent genomic deletion events along their evolution.

scholarly journals Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0253364
Author(s):  
Lee A. Armstrong ◽  
Sven M. Lange ◽  
Virginia Dee Cesare ◽  
Stephen P. Matthews ◽  
Raja Sekhar Nirujogi ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Transmembrane Protein ◽  
Cross Reactivity ◽  
Alternative Methods ◽  
Protease Domain ◽  
Host Cell Proteins ◽  
Active Protease ◽  
Micromolar Range

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

scholarly journals Biochemical Characterization of the Catalytic Domain of Human Matrix Metalloproteinase 19

2000 ◽  
Vol 275 (20) ◽  
pp. 14809-14816 ◽  
Author(s):  
Jan O. Stracke ◽  
Mike Hutton ◽  
Margaret Stewart ◽  
Alberto M. Pendás ◽  
Bryan Smith ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Biochemical Characterization ◽  
Catalytic Domain

Cloning, Overexpression, and Biochemical Characterization of the Catalytic Domain of MutY†

Biochemistry ◽  
1997 ◽  
Vol 36 (37) ◽  
pp. 11140-11152 ◽  
Author(s):  
Raymond C. Manuel ◽  
R. Stephen Lloyd
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Domain

Structural and biochemical characterization of theClostridium perfringensautolysin catalytic domain

FEBS Letters ◽  
2016 ◽  
Vol 591 (1) ◽  
pp. 231-239 ◽  
Author(s):  
Eiji Tamai ◽  
Hiroshi Sekiya ◽  
Eri Goda ◽  
Nahomi Makihata ◽  
Jun Maki ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Domain
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