scholarly journals Biochemical characterization of the catalytic domains of three different clostridial collagenases

2009 ◽  
Vol 390 (1) ◽  
Author(s):  
Ulrich Eckhard ◽  
Esther Schönauer ◽  
Paulina Ducka ◽  
Peter Briza ◽  
Dorota Nüss ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Substrate Affinity ◽  
Catalytic Domains ◽  
Zinc Binding Site ◽  
N Terminus ◽  
Activation Mechanisms ◽  
Order Of Magnitude ◽  
Therapy And Diagnosis ◽  
Prime Target

Abstract Clostridial collagenases are used for a broad spectrum of biotechnological applications and represent prime target candidates for both therapy and diagnosis of clostridial infections. In this study, we biochemically characterized the catalytic domains of three clostridial collagenases, collagenase G (ColG) and H (ColH) from Clostridium histolyticum, and collagenase T (ColT) from C. tetani. All protein samples showed activity against a synthetic peptidic substrate (furylacryloyl-Leu-Gly-Pro-Ala, FALGPA) with ColH showing the highest overall activity and highest substrate affinity. Whereas the K m values of all three enzymes were within the same order of magnitude, the turnover rate k cat of ColG decreased 50- to 150-fold when compared to ColT and ColH. It is noteworthy that the protein N-terminus significantly impacts their substrate affinity and substrate turnover as well as their inhibition profile with 1,10-phenanthroline. These findings were complemented with the discovery of a strictly conserved double-glycine motif, positioned 28 amino acids upstream of the HEXXH zinc binding site, which is critical for enzymatic activity. These observations have consequences with respect to the topology of the N-terminus relative to the active site as well as possible activation mechanisms.

scholarly journals Characterization of four new monoclonal antibodies against the distal N-terminal region of PrPc

2014 ◽  
Author(s):  
Alessandro Didonna ◽  
Anja Colja Venturini ◽  
Katrina Hartman ◽  
Tanja Vranac ◽  
Vladka Curin Serbec ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Prion Protein ◽  
Biochemical Characterization ◽  
Prion Diseases ◽  
Physiological Role ◽  
Prion Infection ◽  
Promising Tool ◽  
C Terminus ◽  
N Terminus

Prion diseases are a group of fatal neurodegenerative disorders that affect humans and animals. They are characterized by the accumulation in the central nervous system of a pathological form of the host-encoded prion protein (PrPC). The prion protein is a membrane glycoprotein that consists of two domains: a globular, structured C-terminus and an unstructured N-terminus. The N-terminal part of the protein is involved in different functions in both health and disease. In the present work we discuss the production and biochemical characterization of a panel of four monoclonal antibodies (mAbs) against the distal N-terminus of PrPC using a well-established methodology based on the immunization of Prnp0/0 mice. Additionally, we show their ability to block prion (PrPSc) replication at nanomolar concentrations in a cell culture model of prion infection. These mAbs represent a promising tool for prion diagnostics and for studying the physiological role of the N-terminal domain of PrPC.

What Can Epitope Specific Antibodies Tell us About the Organization of Caveolin in Cells?

2001 ◽  
Vol 7 (S2) ◽  
pp. 1030-1031
Author(s):  
J.M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Biochemical Characterization ◽  
Cell Types ◽  
Membrane Microdomains ◽  
Truncated Form ◽  
Coated Pits ◽  
N Terminus ◽  
Plasma Membrane Microdomains

There are three members of the caveolin (CAV) gene family that give rise to four polypeptides. These polypeptides are CAV-1α, CAV-1β, CAV-2, and CAV-3. The CAV-1β isoform is a truncated form of CAV-1α that lacks 31 amino acids at the N-terminus of the molecule. The CAV- 1β molecule arises through an alternative splicing mechanism.Caveolae are specialized plasma membrane microdomains that are expressed at high levels in some cell types (e.g., endothelium, adipocytes, fibroblasts). These specialized regions of the plasma membrane have a characteristic omega-shaped appearance with diameters ranging from 40-90 run. They are distinct from clathrin-coated pits since they lack the characteristic coated appearance in electron microscopy. Caveolae were among the first structures to be discovered by biological electron microscopy. However, biochemical characterization of these structures did not begin in earnest until a marker protein was identified. The initial marker was the 22-kDa protein known as caveolin.

scholarly journals Biochemical characterization of a GH70 protein from Lactobacillus kunkeei DSM 12361 with two catalytic domains involving branching sucrase activity

2018 ◽  
Vol 102 (18) ◽  
pp. 7935-7950 ◽  
Author(s):  
Xiangfeng Meng ◽  
Joana Gangoiti ◽  
Xiaofei Wang ◽  
Pieter Grijpstra ◽  
Sander S. van Leeuwen ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Sucrase Activity ◽  
Catalytic Domains ◽  
Lactobacillus Kunkeei

Partial purification and characterization of a non-cyanobacterial cyanophycin synthetase from Acinetobacter calcoaceticus strain ADP1 with regard to substrate specificity, substrate affinity and binding to cyanophycin

Microbiology ◽  
2004 ◽  
Vol 150 (8) ◽  
pp. 2599-2608 ◽  
Author(s):  
Martin Krehenbrink ◽  
Alexander Steinbüchel
Keyword(s):  
Aspartic Acid ◽  
Biochemical Characterization ◽  
Acinetobacter Calcoaceticus ◽  
Partial Purification ◽  
Atp Binding ◽  
Substrate Affinity ◽  
Two Dimensional Gel Electrophoresis ◽  
Atp Binding Site ◽  
Cyanophycin Synthetase

This study reports, for the first time, purification and biochemical characterization of a cyanophycin synthetase from a non-cyanobacterial strain. Cyanophycin synthetase of Acinetobacter calcoaceticus strain ADP1 was purified 69-fold from recombinant Escherichia coli by two chromatographic steps and one novel affinity step utilizing the Mg2+-dependent binding of the enzyme to cyanophycin. Unlike cyanobacterial cyanophycin synthetases characterized so far, the purified enzyme from A. calcoaceticus strain ADP1 did not accept lysine as an alternative substrate to arginine. The apparent K m-values for arginine (47 μM) and aspartic acid (240 μM) were similar to those of known cyanophycin synthetases from cyanobacteria, but this enzyme had a slightly higher affinity for aspartic acid. In addition, the two different ATP-binding sites of the enzyme were characterized independently of each other with respect to K m values for ATP. The ATP-binding site responsible for the addition of arginine was found to have a much higher affinity for ATP (38 μM) than that responsible for the addition of aspartate (210 mM). Furthermore, the binding of the enzyme to the two possible forms of cyanophycin granule polypeptide (CGP), CGP-Asp and CGP-Arg, was studied. While both forms bound around 30–40 % of the enzyme activity present under the assay conditions, binding was Mg2+-dependent in the case of CGP-Asp. Two-dimensional gel electrophoresis revealed that both forms of cyanophycin were equally abundant in cyanophycin-accumulating cells of A. calcoaceticus ADP1.

scholarly journals Structural and Biochemical Characterization of Rm3, a Subclass B3 Metallo-β-Lactamase Identified from a Functional Metagenomic Study

2016 ◽  
Vol 60 (10) ◽  
pp. 5828-5840 ◽  
Author(s):  
Ramya Salimraj ◽  
Lihong Zhang ◽  
Philip Hinchliffe ◽  
Elizabeth M. H. Wellington ◽  
Jürgen Brem ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Distinct Group ◽  
Wide Distribution ◽  
Sequence Comparisons ◽  
Content Type ◽  
Deep Channel ◽  
N Terminus ◽  
Environmental Reservoir ◽  
Metagenomic Study

ABSTRACTβ-Lactamase production increasingly threatens the effectiveness of β-lactams, which remain a mainstay of antimicrobial chemotherapy. New activities emerge through both mutation of previously known β-lactamases and mobilization from environmental reservoirs. The spread of metallo-β-lactamases (MBLs) represents a particular challenge because of their typically broad-spectrum activities encompassing carbapenems, in addition to other β-lactam classes. Increasingly, genomic and metagenomic studies have revealed the distribution of putative MBLs in the environment, but in most cases their activity against clinically relevant β-lactams and, hence, the extent to which they can be considered a resistance reservoir remain uncharacterized. Here we characterize the product of one such gene,blaRm3, identified through functional metagenomic sampling of an environment with high levels of biocide exposure.blaRm3encodes a subclass B3 MBL that, when expressed in a recombinantEscherichia colistrain, is exported to the bacterial periplasm and hydrolyzes clinically used penicillins, cephalosporins, and carbapenems with an efficiency limited by highKmvalues. An Rm3 crystal structure reveals the MBL superfamily αβ/βα fold, which more closely resembles that in mobilized B3 MBLs (AIM-1 and SMB-1) than other chromosomal enzymes (L1 or FEZ-1). A binuclear zinc site sits in a deep channel that is in part defined by a relatively extended N terminus. Structural comparisons suggest that the steric constraints imposed by the N terminus may limit its affinity for β-lactams. Sequence comparisons identify Rm3-like MBLs in numerous other environmental samples and species. Our data suggest that Rm3-like enzymes represent a distinct group of B3 MBLs with a wide distribution and can be considered an environmental reservoir of determinants of β-lactam resistance.

An unusual antithrombin-binding heparin octasaccharide with an additional 3-O-sulfated glucosamine in the active pentasaccharide sequence

2012 ◽  
Vol 449 (2) ◽  
pp. 343-351 ◽  
Author(s):  
Marco Guerrini ◽  
Stefano Elli ◽  
Pierre Mourier ◽  
Timothy R. Rudd ◽  
Davide Gaudesi ◽  
...  
Keyword(s):  
Molecular Conformation ◽  
Biochemical Characterization ◽  
Saturation Transfer ◽  
Antithrombotic Activity ◽  
Saturation Transfer Difference ◽  
Order Of Magnitude ◽  
Epitope Binding ◽  
Docking Energy ◽  
Magnitude Increase

The 3-O-sulfation of N-sulfated glucosamine is the last event in the biosynthesis of heparin/heparan sulfate, giving rise to the antithrombin-binding pentasaccharide sequence AGA*IA, which is largely associated with the antithrombotic activity of these molecules. The aim of the present study was the structural and biochemical characterization of a previously unreported AGA*IA*-containing octasaccharide isolated from the very-low-molecular-mass heparin semuloparin, in which both glucosamine residues of the pentasaccharide moiety located at the non-reducing end bear 3-O-sulfate groups. Two-dimensional and STD (saturation transfer difference) NMR experiments clearly confirmed its structure and identified its ligand epitope binding to antithrombin. The molecular conformation of the octasaccharide–antithrombin complex has been determined by NMR experiments and docking/energy minimization. The presence of the second 3-O-sulfated glucosamine in the octasaccharide induced more than one order of magnitude increase in affinity to antithrombin compared to the pentasaccharide AGA*IA.

scholarly journals Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein

2016 ◽  
Vol 12 (6) ◽  
pp. e1005635 ◽  
Author(s):  
Yaiza Fernández-García ◽  
Juan Reguera ◽  
Carola Busch ◽  
Gregor Witte ◽  
Oliberto Sánchez-Ramos ◽  
...  
Keyword(s):  
Atomic Structure ◽  
Biochemical Characterization ◽  
L Protein ◽  
Andes Virus ◽  
N Terminus ◽  
Rna Endonuclease

scholarly journals Characterization of four new monoclonal antibodies against the distal N-terminal region of PrPc

2014 ◽  
Author(s):  
Alessandro Didonna ◽  
Anja Colja Venturini ◽  
Katrina Hartman ◽  
Tanja Vranac ◽  
Vladka Curin Serbec ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Prion Protein ◽  
Biochemical Characterization ◽  
Prion Diseases ◽  
Physiological Role ◽  
Prion Infection ◽  
Promising Tool ◽  
C Terminus ◽  
N Terminus

Prion diseases are a group of fatal neurodegenerative disorders that affect humans and animals. They are characterized by the accumulation in the central nervous system of a pathological form of the host-encoded prion protein (PrPC). The prion protein is a membrane glycoprotein that consists of two domains: a globular, structured C-terminus and an unstructured N-terminus. The N-terminal part of the protein is involved in different functions in both health and disease. In the present work we discuss the production and biochemical characterization of a panel of four monoclonal antibodies (mAbs) against the distal N-terminus of PrPC using a well-established methodology based on the immunization of Prnp0/0 mice. Additionally, we show their ability to block prion (PrPSc) replication at nanomolar concentrations in a cell culture model of prion infection. These mAbs represent a promising tool for prion diagnostics and for studying the physiological role of the N-terminal domain of PrPC.

scholarly journals Partial purification and biochemical characterization of an extremely thermo- and pH-stable esterase with great substrate affinity

2014 ◽  
Vol 38 ◽  
pp. 538-546 ◽  
Author(s):  
Esra ÖZBEK ◽  
Yakup KOLCUOĞLU ◽  
Leyla KONAK ◽  
Ahmet ÇOLAK ◽  
Fulya ÖZ
Keyword(s):  
Biochemical Characterization ◽  
Partial Purification ◽  
Substrate Affinity

Structural and biochemical characterization of the catalytic domains of GdpP reveals a unified hydrolysis mechanism for the DHH/DHHA1 phosphodiesterase

2018 ◽  
Vol 475 (1) ◽  
pp. 191-205 ◽  
Author(s):  
Feng Wang ◽  
Qing He ◽  
Kaixuan Su ◽  
Tiandi Wei ◽  
Sujuan Xu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Adenosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Catalytic Domains ◽  
Membrane Bound ◽  
Nucleotide Specificity ◽  
Binding Position

The Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterases (PDEs) that catalyze degradation of cyclic di-adenosine monophosphate (c-di-AMP) could be subdivided into two subfamilies based on the final product [5′-phosphadenylyl-adenosine (5′-pApA) or AMP]. In a previous study, we revealed that Rv2837c, a stand-alone DHH/DHHA1 PDE, employs a 5′-pApA internal flipping mechanism to produce AMPs. However, why the membrane-bound DHH/DHHA1 PDE can only degrade c-di-AMP to 5′-pApA remains obscure. Here, we report the crystal structure of the DHH/DHHA1 domain of GdpP (GdpP-C), and structures in complex with c-di-AMP, cyclic di-guanosine monophosphate (c-di-GMP), and 5′-pApA. Structural analysis reveals that GdpP-C binds nucleotide substrates quite differently from how Rv2837c does in terms of substrate-binding position. Accordingly, the nucleotide-binding site of the DHH/DHHA1 PDEs is organized into three (C, G, and R) subsites. For GdpP-C, in the C and G sites c-di-AMP binds and degrades into 5′-pApA, and its G site determines nucleotide specificity. To further degrade into AMPs, 5′-pApA must slide into the C and R sites for flipping and hydrolysis as in Rv2837c. Subsequent mutagenesis and enzymatic studies of GdpP-C and Rv2837c uncover the complete flipping process and reveal a unified catalytic mechanism for members of both DHH/DHHA1 PDE subfamilies.

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