Biochemical Characterization of a Multidomain Deubiquitinating Enzyme Ubp15 and the Regulatory Role of Its Terminal Domains

Biochemistry ◽  
2011 ◽  
Vol 50 (29) ◽  
pp. 6423-6432 ◽  
Author(s):  
William P. Bozza ◽  
Zhihao Zhuang
Keyword(s):  
Biochemical Characterization ◽  
Regulatory Role ◽  
Deubiquitinating Enzyme ◽  
Terminal Domains

scholarly journals Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1

2020 ◽  
Vol 75 (9) ◽  
pp. 2554-2563 ◽  
Author(s):  
Christopher Fröhlich ◽  
Vidar Sørum ◽  
Sandra Huber ◽  
Ørjan Samuelsen ◽  
Fanny Berglund ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Homology Modelling ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
Human Pathogens ◽  
E Coli ◽  
X Ray Crystallography ◽  
Environmental Reservoir

Abstract Background MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure–activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure. Objectives To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1. Methods The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1). Results Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity. Conclusions These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.

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.

Biochemistry and site-directed mutational analysis of Δ7-sterol-C5(6)-desaturase

2000 ◽  
Vol 28 (6) ◽  
pp. 799-803 ◽  
Author(s):  
A. Rahier ◽  
P. Benveniste ◽  
T. Husselstein ◽  
M. Taton
Keyword(s):  
Biochemical Characterization ◽  
Mutational Analysis ◽  
Enzyme System ◽  
Conserved Residues ◽  
Catalytic Role ◽  
Membrane Bound ◽  
Haem Iron ◽  
Oxygen Site

This report describes recent work on the process of desaturation at C5(6) of sterol precursors in plants. Biochemical characterization of the plant Δ7-sterol C5(6)-desaturase (5-DES) indicates that the enzyme system involved shows important similarities to the soluble and membrane-bound non-haem iron desaturases found in eukaryotes, including cyanide and hydrophobic chelators sensitivity, CO resistance and a requirement for exogenous reductant and molecular oxygen. Site-directed mutational analysis of highly conserved residues in 5-DES indicated that eight histidine residues from three histidine-rich motifs were essential for the catalysis, possibly by providing the ligands for a putative Fe centre. This mutational analysis also revealed the catalytic role of the functionally conserved Thr-114.

scholarly journals Functional and Structural Characterization of a Novel Isoamylase from Ostreococcus tauri and Role of the N-Terminal Domain

2020 ◽  
Vol 14 (1) ◽  
pp. 1-11
Author(s):  
Nicolas Hedín ◽  
Julieta Barchiesi ◽  
Diego F. Gomez-Casati ◽  
María V. Busi
Keyword(s):  
Crystallization Process ◽  
Biochemical Characterization ◽  
Carbohydrate Binding ◽  
Amino Acid Residues ◽  
Photosynthetic Organisms ◽  
Ostreococcus Tauri ◽  
The Family ◽  
Almost All

Background: The debranching starch enzymes, isoamylase 1 and 2 are well-conserved enzymes present in almost all the photosynthetic organisms. These enzymes are involved in the crystallization process of starch and are key components which remove misplaced α-1,6 ramifications on the final molecule. Aim: In this work, we performed a functional and structural study of a novel isoamylase from Ostreococcus tauri. Methods: We identified conserved amino acid residues possibly involved in catalysis. We also identified a region at the N-terminal end that resembles a Carbohydrate Binding Domain (CBM), which is more related to the family CBM48, but has no spatial conservation of the residues involved in carbohydrate binding. Results: The cloning, expression and biochemical characterization of this N-terminal region confirmed that it binds to polysaccharides, showing greater capacity for binding to amylopectin rather than total starch or amylose. Conclusion: This module could be a variant of the CBM48 family or it could be classified within a new CBM family.

scholarly journals Discovery and characterization of a novel family of prokaryotic nanocompartments involved in sulfur metabolism

2020 ◽  
Author(s):  
Robert J. Nichols ◽  
Benjamin LaFrance ◽  
Naiya R. Phillips ◽  
Luke M. Oltrogge ◽  
Luis E. Valentin-Alvarado ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Biochemical Characterization ◽  
Sulfur Metabolism ◽  
Physiological Role ◽  
Enzymatic Characterization ◽  
Cysteine Desulfurase ◽  
Future Studies ◽  
Pcc 7942

AbstractProkaryotic nanocompartments, also known as encapsulins, are a recently discovered proteinaceous organelle in prokaryotes that compartmentalize cargo enzymes. While initial studies have begun to elucidate the structure and physiological roles of encapsulins, bioinformatic evidence suggests that a great diversity of encapsulin nanocompartments remains unexplored. Here, we describe a novel encapsulin in the freshwater cyanobacterium Synechococcus elongatus PCC 7942. This nanocompartment is upregulated upon sulfate starvation and encapsulates a cysteine desulfurase enzyme via an N-terminal targeting sequence. Using cryoelectron microscopy, we have determined the structure of the nanocompartment complex to 2.2 Å resolution. Lastly, biochemical characterization of the complex demonstrated that the activity of the cysteine desulfurase is enhanced upon encapsulation. Taken together, our discovery, structural analysis, and enzymatic characterization of this prokaryotic nanocompartment provide a foundation for future studies seeking to understand the physiological role of this encapsulin in various bacteria.

Biochemical characterization of laccase from hairy root culture of Brassica juncea L. and role of redox mediators to enhance its potential for the decolorization of textile dyes

Planta ◽  
2011 ◽  
Vol 234 (6) ◽  
pp. 1137-1149 ◽  
Author(s):  
Amar A. Telke ◽  
Anuradha N. Kagalkar ◽  
Umesh B. Jagtap ◽  
Neetin S. Desai ◽  
Vishwas A. Bapat ◽  
...  
Keyword(s):  
Brassica Juncea ◽  
Hairy Root ◽  
Biochemical Characterization ◽  
Root Culture ◽  
Hairy Root Culture ◽  
Textile Dyes ◽  
Redox Mediators
Sign in / Sign up

Export Citation Format

Share Document