scholarly journals The catalytic domain of the germination-specific lytic transglycosylase SleB fromBacillus anthracisdisplays a unique active site topology

2012 ◽  
Vol 80 (10) ◽  
pp. 2469-2475 ◽  
Author(s):  
Xing Jing ◽  
Howard R. Robinson ◽  
Jared D. Heffron ◽  
David L. Popham ◽  
Florian D. Schubot
Keyword(s):  
Active Site ◽  
Catalytic Domain ◽  
Lytic Transglycosylase

scholarly journals The Catalytic Domain ofEscherichia coliLon Protease Has a Unique Fold and a Ser-Lys Dyad in the Active Site

2003 ◽  
Vol 279 (9) ◽  
pp. 8140-8148 ◽  
Author(s):  
Istvan Botos ◽  
Edward E. Melnikov ◽  
Scott Cherry ◽  
Joseph E. Tropea ◽  
Anna G. Khalatova ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Domain

Crystal structure of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis

2021 ◽  
Vol 77 (11) ◽  
Author(s):  
Kohei Sasamoto ◽  
Tomoki Himiyama ◽  
Kunihiko Moriyoshi ◽  
Takashi Ohmoto ◽  
Koichi Uegaki ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cellulose Acetate ◽  
Electron Density ◽  
Active Site ◽  
Catalytic Domain ◽  
Crystal Packing ◽  
Signal Sequence ◽  
Industrial Applications ◽  
Side Chain ◽  
Active Site Conformation

The acetylxylan esterases (AXEs) classified into carbohydrate esterase family 4 (CE4) are metalloenzymes that catalyze the deacetylation of acetylated carbohydrates. AXE from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866), which belongs to CE4, is composed of three parts: a signal sequence (residues 1–22), an N-terminal region (NTR; residues 23–135) and a catalytic domain (residues 136–324). TTE0866 catalyzes the deacetylation of highly substituted cellulose acetate and is expected to be useful for industrial applications in the reuse of resources. In this study, the crystal structure of TTE0866 (residues 23–324) was successfully determined. The crystal diffracted to 1.9 Å resolution and belonged to space group I212121. The catalytic domain (residues 136–321) exhibited a (β/α)7-barrel topology. However, electron density was not observed for the NTR (residues 23–135). The crystal packing revealed the presence of an intermolecular space without observable electron density, indicating that the NTR occupies this space without a defined conformation or was truncated during the crystallization process. Although the active-site conformation of TTE0866 was found to be highly similar to those of other CE4 enzymes, the orientation of its Trp264 side chain near the active site was clearly distinct. The unique orientation of the Trp264 side chain formed a different-shaped cavity within TTE0866, which may contribute to its reactivity towards highly substituted cellulose acetate.

Structural insights into dihydroxylation of terephthalate, a product of polyethylene terephthalate degradation

2022 ◽  
Author(s):  
Jai Krishna Mahto ◽  
Neetu Neetu ◽  
Monica Sharma ◽  
Monika Dubey ◽  
Bhanu Prakash Vellanki ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Polyethylene Terephthalate ◽  
Active Site ◽  
Catalytic Domain ◽  
Structural Features ◽  
Comamonas Testosteroni ◽  
Plastic Pollution ◽  
Microbial Utilization ◽  
Steady State Kinetics

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

scholarly journals PENENTUAN SISI AKTIF SELULASE ASPERGILLUS NIGER DENGAN DOCKING LIGAN

2014 ◽  
Vol 16 (2) ◽  
pp. 94-100 ◽  
Author(s):  
Tigor Nauli
Keyword(s):  
Aspergillus Niger ◽  
Active Site ◽  
Molecular Conformation ◽  
Catalytic Domain ◽  
Binding Free Energy ◽  
Docking Simulation ◽  
Optimization Approach ◽  
Calculation Results ◽  
Substrate Binding Domain ◽  
Specific Metal

Letak dari sisi aktif selulase Aspergillus niger, yang akan menentukan aktivitas katalitiknya, dapat diketahui melalui komputasi. Sebuah ligan selobiosa dimodelkan untuk dapat melakukan simulasi docking pada molekul selulase yang telah diketahui struktur kristalnya. Melalui kalkulasi energi ikatan dan pendekatan optimasi memakai algoritma genetik Lamarckian, dapat dipilih konformasi molekul yang menunjukkan adanya daerah tertentu dengan energi terendah. Struktur yang memiliki daerah semacam ini dianggap mewakili konfigurasi terbaik terikatnya ligan pada sisi aktif yang dicari.Hasil perhitungan memperlihatkan bahwa tekukan protein yang membentuk celah konkaf diantara dua kelompok struktur b-sheet yang saling berlawanan arah pada molekul selulase merupakan sisi aktif dari enzim tersebut. Ligan dapat terikat disana melalui interaksi hidrofilik dengan residu asparagin (Asn20), serin (Ser111), dan glutamin (Gln158). Di salah satu ujung sisi aktif selulase terdapat residu aspartat (Asp99) dan glutamat (Glu116, Glu204) yang akan mempengaruhi aksi katalitik dari enzim selulase apabila residu-residu ini terikat oleh ion-ion divalen.Sisi aktif selulase ini merupakan gabungan dari domain pengikat substrat dan domain katalitik. Penambahan ion logam yang tepat pada sisi aktif enzim selulase Aspergillus niger dapat meningkatkan aktivitas spesifiknya.Kata kunci:docking, ligan, selulase, sisi ikatan, substrat The active site of cellulase from Aspergillus niger that affects the enzyme activity can be searched by computational methods. A ligand of cellobiose is modelled to perform docking simulation to cellulase with known crystal structure. By calculating the binding free energy and optimization approach using Lamarckian's genetic algorithm, a molecular conformation that has a region with the lowest energy value can be selected. The molecule structure with such region represents the best configuration of ligand bound to the active site.The calculation results that the concave cleft formed by protein folding of two anti-parallel b-sheet structures is the active site of the enzyme. A ligand would bind to the site through hydrophilic interactions with asparagine (Asn20), serine (Ser111), and glutamine (Gln158) residues. The aspartic acid (Asp99) and glutamic acid (Glu116, Glu204) residues that reside in one end of the active site determine the catalytic actions of the enzyme when they are binding with some metal ions.It is shown that the active site of this cellulase has substrate-binding domain and catalytic domain together. The introduction of specific metal ions to the active site of Asperillus niger cellulase will increase its specific activity.Keywords: binding site, cellulase, docking, ligand, substrate

scholarly journals A trapped human PPM1A–phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity

2018 ◽  
Vol 293 (21) ◽  
pp. 7993-8008 ◽  
Author(s):  
Subrata Debnath ◽  
Dalibor Kosek ◽  
Harichandra D. Tagad ◽  
Stewart R. Durell ◽  
Daniel H. Appella ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Metal Ions ◽  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Catalytic Domain ◽  
Protein Phosphatases ◽  
Random Order ◽  
Structural Features

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro. The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E–c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.

scholarly journals Cwp19 Is a Novel Lytic Transglycosylase Involved in Stationary-Phase Autolysis Resulting in Toxin Release inClostridium difficile

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Sandra Wydau-Dematteis ◽  
Imane El Meouche ◽  
Pascal Courtin ◽  
Audrey Hamiot ◽  
René Lai-Kuen ◽  
...  
Keyword(s):  
Health Care ◽  
Clostridium Difficile ◽  
Stationary Phase ◽  
Catalytic Domain ◽  
Brain Heart Infusion ◽  
Surface Protein ◽  
Wild Type ◽  
Content Type ◽  
Lytic Transglycosylase ◽  
Cell Autolysis

ABSTRACTClostridium difficileis the major etiologic agent of antibiotic-associated intestinal disease. Pathogenesis ofC. difficileis mainly attributed to the production and secretion of toxins A and B. Unlike most clostridial toxins, toxins A and B have no signal peptide, and they are therefore secreted by unusual mechanisms involving the holin-like TcdE protein and/or autolysis. In this study, we characterized the cell surface protein Cwp19, a newly identified peptidoglycan-degrading enzyme containing a novel catalytic domain. We purified a recombinant His6-tagged Cwp19 protein and showed that it has lytic transglycosylase activity. Moreover, we observed that Cwp19 is involved in cell autolysis and that aC. difficilecwp19mutant exhibited delayed autolysis in stationary phase compared to the wild type when bacteria were grown in brain heart infusion (BHI) medium. Wild-type cell autolysis is correlated to strong alterations of cell wall thickness and integrity and to release of cytoplasmic material. Furthermore, we demonstrated that toxins were released into the extracellular medium as a result of Cwp19-induced autolysis when cells were grown in BHI medium. In contrast, Cwp19 did not induce autolysis or toxin release when cells were grown in tryptone-yeast extract (TY) medium. These data provide evidence for the first time that TcdE and bacteriolysis are coexisting mechanisms for toxin release, with their relative contributionsin vitrodepending on growth conditions. Thus, Cwp19 is an important surface protein involved in autolysis of vegetative cells ofC. difficilethat mediates the release of the toxins from the cell cytosol in response to specific environment conditions.IMPORTANCEClostridium difficile-associated disease is mainly known as a health care-associated infection. It represents the most problematic hospital-acquired infection in North America and Europe and exerts significant economic pressure on health care systems. Virulent strains ofC. difficilegenerally produce two toxins that have been identified as the major virulence factors. The mechanism for release of these toxins from bacterial cells is not yet fully understood but is thought to be partly mediated by bacteriolysis. Here we identify a novel peptidoglycan hydrolase inC. difficile, Cwp19, exhibiting lytic transglycosylase activity. We show that Cwp19 contributes toC. difficilecell autolysis in the stationary phase and, consequently, to toxin release, most probably as a response to environmental conditions such as nutritional signals. These data highlight that Cwp19 constitutes a promising target for the development of new preventive and curative strategies.

scholarly journals Crystal structure of full‐length human collagenase 3 (MMP‐13) with peptides in the active site defines exosites in the catalytic domain

2013 ◽  
Vol 27 (11) ◽  
pp. 4395-4405 ◽  
Author(s):  
Enrico A. Stura ◽  
Robert Visse ◽  
Philippe Cuniasse ◽  
Vincent Dive ◽  
Hideaki Nagase
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Catalytic Domain ◽  
Full Length

scholarly journals Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR

2013 ◽  
Vol 288 (23) ◽  
pp. 17008-17018 ◽  
Author(s):  
D. Fernando Estrada ◽  
Jennifer S. Laurence ◽  
Emily E. Scott
Keyword(s):  
Cytochrome P450 ◽  
Binding Site ◽  
Active Site ◽  
Catalytic Domain ◽  
Cytochrome B5 ◽  
Structural Basis ◽  
Reaction Conditions ◽  
Reversible Binding ◽  
Important Interaction

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.

Evidence for a Novel Splice Variant of the Gene for Platelet Activating Factor Acetylhydrolase 1B2 (PAFAH1B2), an Important Signal Terminator in the Platelet Activating Factor (PAF) Pathway.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2630-2630
Author(s):  
Nels Olson ◽  
Bruce Scott ◽  
George Long ◽  
Edwin Bovill
Keyword(s):  
Alternative Splice ◽  
Active Site ◽  
Splice Variant ◽  
Catalytic Domain ◽  
Platelet Activating Factor ◽  
Type I ◽  
Native Form ◽  
Western Blots ◽  
Intracellular Form ◽  
A Genome

Abstract Introduction: A genome-wide scan of linkage with venous thrombosis in a thrombophilic kindred with type I protein C deficiency identified a candidate region on chromosome 11q23 (P<0.0001). Located at the linkage peak is PAFAH1B2, the β-subunit of the intracellular form of PAFAH. Intracellular PAFAH is a heterodimer of alpha and beta subunits both of which have catalytic activity. PAFAH reduces intracellular PAF levels and a reduction in active PAFAH likely leads to an enhanced inflammatory response at the vascular wall. Resequencing experiments revealed a novel splice variant for the intracellular form of PAFAH. The current study characterizes this novel splice variant that results in the deletion of exon 6 and its catalytic domain and the introduction of an alternative 7th exon. Methods: Identification of novel mRNA splice variants was achieved by sequencing PCR products obtained from amplifying known regions of PAFAH 1B2 mRNA. Confirmation of the exon 5 to 7 splice and exon 7 polyadenylation were obtained from sequencing 3′RACE products. Protein extracts were resolved by SDS- PAGE and Western blotted. Results: An alternative splice form of PAFAH 1B2 was isolated and sequenced. This isoform was observed in cDNA from human liver, moncytes, and T and B lymphocytes using PCR. This variant contains the first five exons of the native form protein and replaces exon 6 with an alternative 7th exon located downstream of exon 6. Exon 6 contains two residues involved in the protein’s active site triad, Asp and His, and are absent in exon 7. Computer modeling suggests replacement of exon 6 by exon 7 would abolish the active site and possibly alter the proteins’ ability to dimerize. Preliminary Western blots from selected tissues demonstrated the presence in liver of a new band consistent with the predicted size of the variant. Conclusions: We hypothesize that this alternative splice likely decreases the proteins ability to function as a regulator of inflammation. We are presently carrying out gene expression analysis to determine the relative levels of the splice variant in different tissues and changes in expression associated with activation of leukocytes.

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